CN111217904A

CN111217904A - Preparation method of polymerized hemoglobin with low high polymer content

Info

Publication number: CN111217904A
Application number: CN202010198827.3A
Authority: CN
Inventors: 游可为; 史国营; 张彦鹏; 石松; 陈浩源
Original assignee: Runfang Beijing Biotechnology Co Ltd; Redpharm Beijing Biomedical Research Institute Co ltd
Current assignee: Redpharm Beijing Biomedical Research Institute Co ltd; Runfang Beijing Biotechnology Co ltd
Priority date: 2019-09-09
Filing date: 2020-03-20
Publication date: 2020-06-02
Anticipated expiration: 2040-03-20
Also published as: CN110563836A; CN111217904B

Abstract

The invention relates to a preparation method of polymerized hemoglobin with low high polymer content, and particularly provides a preparation method of cross-linked hemoglobin, which comprises the following steps: a. diluting the deoxygenated hemoglobin; b. the cross-linking agent glutaraldehyde and the diluted deoxyhemoglobin are subjected to cross-linking reaction; c. adding a reducing agent to terminate the crosslinking reaction, and removing unreacted crosslinking agent and terminating the reducing agent by an ultrafiltration liquid exchange mode to obtain crosslinked hemoglobin; wherein: in the step a, the concentration of the deoxyhemoglobin is diluted to 1-3g/dL, and N-acetyl-L-cysteine is added into the hemoglobin, so that the mass ratio of the hemoglobin to the N-acetyl-L-cysteine in the solution is 5-10: 1; and the mass ratio of the hemoglobin to the cross-linking agent is 1000: 29-45. The polymerized hemoglobin prepared by the method has low dimer content and low high polymer content.

Description

Preparation method of polymerized hemoglobin with low high polymer content

PRIORITY INFORMATION

The present patent application claims priority from chinese patent application 201910846580.9 entitled "method for preparing cross-linked hemoglobin and organ perfusate containing the hemoglobin" filed as 2019, 9.9.9, which is incorporated herein by reference in its entirety.

Technical Field

The invention belongs to the field of biological pharmacy, relates to a polymerization method of hemoglobin, and particularly relates to a preparation method of polymerized hemoglobin with low high polymer content.

Background

Blood transfusion is an essential medical tool for surgery, emergency and disaster resistance, and battlefield rescue. In recent years, worldwide demand for blood for transfusion has increased geometrically. However, because the blood type of human blood is very complex, the transfusion reaction is difficult to overcome all the time, the storage and transportation of natural blood are very difficult, and in addition, due to the serious pollution of viruses such as HBV and HIV to blood, the number of blood donors is seriously insufficient, and the like, the safe and effective blood source is increasingly in short supply. Therefore, the substitute for red blood cells has become a research hotspot of international interest and is listed as a major research and development field of twenty-first century.

the ideal erythrocyte substitute is required to have Oxygen transfer function, biocompatibility, safety and stability of natural erythrocyte, i.e. ② has high Oxygen carrying capacity, Oxygen partial pressure in normal physiological range and can effectively supply Oxygen to tissues, ② has good biocompatibility with all components of human blood, ⑦ can maintain osmotic pressure, acid-base balance, viscosity and blood volume, ② has no immunogenicity (no allergenicity), no pollution of blood pathogenic microorganism and no pyrogen, the half life of internal circulation of the substitute is over 24h, no side effects such as nephrotoxicity and the like under normal perfusion condition, the product shelf life is more than 6 months under low temperature condition, and ② has expected removability.

Perfluorocarbons are synthetic compounds with high oxygen carrying capacity that dissolve 40-50% by volume of oxygen at 21.3KPa (160mmHg) oxygen partial pressure and 37 ℃. Although perfluorocarbons are insoluble in water, they can be emulsified with surfactants for infusion. 20% perfluorodecalin (Flousol-DA20TM), produced by osaka green cross, japan, is an oxygen-carrying volume expander currently approved by the FDA for use in the united states for coronary perfusion after coronary angioplasty. The high oxygen solubility of perfluorocarbons is significant in the treatment of dyspnea syndrome. However, oxygen carried by perfluorocarbons is released immediately and is difficult to carry to certain anoxic tissues, meanwhile, oxygen is accumulated in partial tissues to cause tissue damage, and the perfluorocarbons have the defects of inconvenient storage, slow metabolism in vivo and side effects similar to cold symptoms, are not as good in biocompatibility and oxygen carrying capacity as hemoglobin, are not completely guaranteed in long-term use effect and safety, and therefore have certain limitations in application.

Hemoglobin is a biomolecule that carries oxygen in a living body, and is present in erythrocytes in blood of animals such as humans, cows, horses, and the like. Because the protein is protein, the blood matching type is not needed, and the red blood cell oxygen carrier is easy to prepare and store for a long time and has the potential of replacing the oxygen carrying function of red blood cells.

Hemoglobin is one of the constituents of red blood cells, and has functions of high-efficiency oxygen carrying capacity and maintaining colloid osmotic pressure. Hemoglobin from a wide variety of sources, including human, animal, recombinant hemoglobin, and the like, can be prepared as erythroblast substitutes by crosslinking, conjugation, polymerization, and the like. The primary purpose of these modifications is to prolong the retention time of hemoglobin in the blood circulation, reduce toxicity and ensure oxygen release to the tissues. At present, the research and development of the red blood cell substitute taking hemoglobin as a matrix is mainly directed in various countries.

The free hemoglobin is used as raw material to develop the substitute of erythrocyte, and the hemoglobin must be modified to meet the physiological requirement in human body. Chemical modification of hemoglobin is one of research approaches, and hemoglobin molecules are modified at the molecular level by chemical methods, i.e., in vitro, the active group of hemoglobin is covalently linked with some chemical groups, especially macromolecules with biocompatibility, by artificial methods, so as to change the properties of hemoglobin, namely, the chemical modification of hemoglobin.

Surface-modified hemoglobins are the conjugation products of hemoglobin molecules and macromolecules (e.g., dextran, polyethylene glycol, dextran). The primary purpose of surface modification of hemoglobin is to extend the residence time of hemoglobin in blood vessels.

Chang reported in 1964 that hemoglobin crosslinked with polymers could form an insoluble coupled hemoglobin. Later Wong improved this technique by cross-linking dextran with hemoglobin to form soluble coupled hemoglobin. The company Sangart, USA, developed Hemospan (MP4, polyethylene glycol (PEG) modified human hemoglobin) as a new generation of red blood cell substitute. The interaction force of PEG and water is strong, so that a layer of water protective film is formed on the surface of hemoglobin, the immunogenicity can be eliminated, the effective molecular radius can be increased, the half-life period in vivo can be prolonged, the viscosity similar to that of blood can be provided, the vasoconstriction can be prevented, and the normal oxygen conveying and releasing capacity can be maintained.

intramolecular cross-linking of hemoglobin refers to the formation of relatively stable covalent bonds between hemoglobin subunits such as the alpha and beta subunits, α β and α β, using small molecule substances to stabilize the hemoglobin molecule 1968, bond and Jandl reported that a bifunctional reagent bis (N-maleimidomethyl) capable of intramolecular cross-linking, which reacts with hemoglobin to form a cross-linked tetramer to prevent dissociation of the tetrameric hemoglobin molecule and thereby prolong its residence time in blood vessels.

Polymerized hemoglobin is a stable polymerization product of hemoglobin molecules polymerized using a bifunctional reagent such as glutaraldehyde to form a polymerized hemoglobin molecule. The bifunctional cross-linking reagent comprises glutaraldehyde, and Chang firstly uses the bifunctional cross-linking reagent to cross-link reactive amino acid residues of hemoglobin to obtain the polymerized hemoglobin. PolyHeme, a product of Northfield, is prepared by first intramolecular cross-linking of human hemoglobin using pyridoxal, followed by further intermolecular cross-linking with glutaraldehyde, resulting in a larger molecular weight of polymerized hemoglobin. Hemopure (TM), manufactured by Biopure corporation, cross-links purified bovine hemoglobin using glutaraldehyde to form polymerized bovine hemoglobin, which has passed clinical trials in south Africa and is on the market.

It is known that polymerized hemoglobin with glutaraldehyde as a cross-linking agent is the most studied product in red blood cell substitutes, the earliest entering phase III clinics and being marketed in some countries. The preparation process of glutaraldehyde polymeric hemoglobin can be roughly divided into the following steps: crude purification of hemoglobin, fine purification of hemoglobin, polymerization (modification) of hemoglobin. Among these three steps, the polymerization process of hemoglobin is important. Because while glutaraldehyde is one of the most commonly used homobifunctional cross-linking agents for the preparation of polymerized hemoglobin, glutaraldehyde also has a number of disadvantages: it has two aldehyde groups, so the reactivity is very high; it has long carbon chains, which facilitates intermolecular crosslinking, resulting in excessive molecular weight of the polymerized hemoglobin; without specific reactive sites, about 40 lysine side chain amino groups and four chain end amino groups on the surface of hemoglobin can participate in the reaction, making the polymerization difficult to control. The above-mentioned disadvantages of glutaraldehyde are finally manifested by varying sizes of the polymerized hemoglobin molecules in solution, with molecular weight distributions ranging from 32kD to over 1000 kD. The wide molecular weight distribution not only reduces the effective utilization rate of hemoglobin, but also has poor stability of too low molecular weight, easy dissociation to cause renal toxicity and low oxygen carrying efficiency, and in addition, the content of high polymer polymerized hemoglobin is increased. While a high average molecular weight does not alter the oxygen-carrying properties of the polymerized hemoglobin, it reduces the plasma oncotic pressure and increases viscosity, which in turn affects its hemodynamic properties and tissue oxygenation capacity. Therefore, the U.S. food and drug administration has specifically requested a reduction in the average molecular weight in a batch reply to the Biopure phase III clinical trial of glutaraldehyde polymerization of bovine hemoglobin.

Therefore, the polymerization process of hemoglobin is not only a key of the whole process, but also a bottleneck restricting the commercialization of the polymerized hemoglobin.

Disclosure of Invention

In order to solve the above problems, the inventors have surprisingly found, through extensive experimental investigations, that the dilution of hemoglobin The release concentration, the mass ratio of hemoglobin to glutaraldehyde, and the mass ratio of hemoglobin to N-acetyl-L-cysteine (NAC) were prepared About three important factors of the polymerization process, in particular:

first, the inventors found that the diluted concentration of hemoglobin should be controlled to 1-3g/dL If the content is more than 3g/dL, the content of the high polymer in the polymerized hemoglobin is too high; the dilution concentration of hemoglobin is less than 1 g- dL, although the molecular weight distribution of the polymerized hemoglobin is within the acceptable range, a greater dilution is required to account for the lower dilution concentration The polymerization volume consumes more production cost, and the lower dilution concentration is not recommended. Thus, hemoglobin and glutaraldehyde The dilute concentration of hemoglobin has a significant effect on the molecular weight distribution of the polymerized hemoglobin.

Second, the inventors found thatThe mass ratio of the hemoglobin to the glutaraldehyde is controlled to be 1000:29-45 of hemoglobin and if the mass ratio of the glutaraldehyde is less than 1000:45, i.e., too high a glutaraldehyde addition, will result in the inclusion of high polymers in the polymerized hemoglobin Too high an amount; if the mass ratio of the hemoglobin to the glutaraldehyde is more than 1000:29, i.e., too low an amount of glutaraldehyde added, will result in polymerized blood The content of dimers in the hemoglobin is too high. Thus, in the polymerization process of hemoglobin and glutaraldehyde, the polymerization process of hemoglobin and glutaraldehyde The mass ratio has a significant effect on the molecular weight distribution of the polymerized hemoglobin.

Third, 1) the inventors found that before polymerization of hemoglobin with glutaraldehyde, the addition of NAC was compared to the absence of NAC After NAC, the content of dimer in the polymerized hemoglobin can be obviously reduced, and the polymerized hemoglobin can be obviously reduced The content of the high polymer exerts a new function different from the conventional reduction action of the high polymer, and obtains unexpected technical effects; 2) the inventors have found that the addition of other conventional reducing agents (such as glutathione) prior to the polymerization of hemoglobin with glutaraldehyde Peptide, mercaptoethylamine), the content of dimer and high polymer in the polymerized hemoglobin is obviously reduced when NAC is added, and the method obtains An unexpected technical effect; 3) the inventors have found that hemoglobin can be reacted with glutaraldehyde before polymerization of the hemoglobin The mass ratio of the NAC to be added is controlled to be 5-10:1, if the mass ratio of the hemoglobin to the NAC is more than 5-10:1, such as 100: 1 (this) Hemoglobin to NAC molar ratio of about 0.25: 1) this results in a high dimer content in the polymerized hemoglobin. Therefore, the temperature of the molten metal is controlled, in the polymerization process of hemoglobin and glutaraldehyde, whether NAC is added or not and the mass ratio of hemoglobin to NAC The molecular weight distribution of the protein has a significant effect.

In conclusion, the inventor finds that the dilution concentration of the hemoglobin is controlled to be 1-3g/dL, and the hemoglobin and glutaraldehyde are Controlling the quantity ratio at 1000:29-45, and controlling the mass ratio of the hemoglobin to the N-acetyl-L-cysteine (NAC) to be 5-10: when the pressure of the mixture is 1, the pressure is lower, the polymerized hemoglobin has low content of dimerThe content of the high polymer is also lower, thereby achieving unexpected skill The operation effect makes a substantial contribution to the prior art.

In addition, it is specifically stated that claims 1 to 2 of the present application are derived from priority document 201910846580.9 The contents of the description are explicitly described in the present specification without departing from the scope of the description of the priority documents The specific correspondence is shown in the following table:

to this end, in a first aspect of the present invention, there is provided a method for preparing cross-linked hemoglobin, comprising the steps of:

a. diluting deoxygenated hemoglobin to X₁(ii) a Adding to said diluted deoxygenated hemoglobin a final concentration of X₂Obtaining reaction liquid by the N-acetyl-L-cysteine; transferring the reaction liquid into an oxygen-free reaction container;

b. fixing a pressure atomizer at the top of the anaerobic reaction vessel, connecting an air inlet pipe and a liquid inlet pipe to the atomizer, and connecting a balance air pipeline at the top of the anaerobic reaction vessel; closing the anaerobic reaction vessel, starting a stirring rotor at the bottom, and heating the reaction solution to 42 ℃ through a water bath jacket; opening the valve of the liquid inlet pipe, adding the cross-linking agent glutaraldehyde, and adjusting the liquid inlet speed to X₃Opening a gas inlet pipe valve, introducing inert gas, preferably high-purity nitrogen, and regulating and controlling a gas outlet valve on the balance gas pipeline to balance the pressure of the anaerobic reaction container to X₄According to 1g of hemoglobin X₅Adding the cross-linking agent glutaraldehyde into the reaction solution according to the proportion of glutaraldehyde to carry out cross-linking reaction;

c. adding sodium borohydride to terminate the crosslinking reaction, and purifying the 30kD ultrafiltration exchange liquid to obtain the crosslinked hemoglobin;

wherein, X is₁、X₂、X₃、X₄And X₅Selected from one of the following combinations:

in a second aspect of the present invention, there is provided a method for preparing cross-linked hemoglobin, comprising the steps of:

a. diluting the deoxygenated hemoglobin to 2 g/dL; adding N-acetyl-L-cysteine with the final concentration of 3mg/mL into the diluted and deoxidized hemoglobin to obtain a reaction solution; transferring the reaction liquid into an oxygen-free reaction container;

b. closing the anaerobic reaction vessel, starting a stirring rotor at the bottom, and heating the reaction solution to 42 ℃ through a water bath jacket; placing a static mixer in a circulating pipeline of a reaction system, arranging a branch pipeline at the static mixer, adding a cross-linking agent glutaraldehyde, fully mixing the hemoglobin and the cross-linking agent at the static mixer, introducing inert gas, preferably high-purity nitrogen into the oxygen-free reaction vessel at the cross-linking agent adding speed of 100mL/min, and adding the cross-linking agent glutaraldehyde into the reaction liquid according to the proportion of 1g of hemoglobin to 37mg of glutaraldehyde for carrying out cross-linking reaction;

c. and (3) adding sodium borohydride to terminate the crosslinking reaction, and purifying the 30kD ultrafiltration exchange liquid to obtain the crosslinked hemoglobin.

In a third aspect of the invention, the invention provides an organ perfusion fluid comprising: cross-linked hemoglobin prepared by the foregoing method; vitamin K1; and prostacyclin.

In some embodiments, the organ perfusate comprises:

10-50g of cross-linked hemoglobin prepared by the above method

Human albumin (Shandongtai bang biology, pharmaceutical grade) 40-60g

Heparin (Chenxin pharmaceutical industry) 8000-12000IU

8.4% sodium bicarbonate (Tiandi pharmacy) 8-12mL

8-12mL of 10% calcium gluconate (Huarun double crane)

Vancomycin (North China pharmaceutical) 0.4-0.6mg

Gentamicin (North China pharmaceutical) 0.05-0.07mg

40-60mL of 10% compound amino acid injection (Huarui pharmaceutical)

0.1-0.3mL of 12 kinds of compound vitamins for injection (Pude pharmaceutical industry)

Vitamin K1 (honest pharmaceutical industry) 0.05-0.15mg

2ug/mL prostacyclin (Mingren medicine) 8-32mL

The content of each component refers to the content in 1000mL of the organ perfusate.

In a fourth aspect of the present invention, there is provided a method for preparing cross-linked hemoglobin, comprising the steps of:

a1, diluting the deoxyhemoglobin, and adding the diluted deoxyhemoglobin into an anaerobic reactor;

b1, arranging an atomizer at the top of the anaerobic reactor, and adding a cross-linking agent into the reactor through the atomizer to perform a cross-linking reaction;

c1, adding a reducing agent to terminate the crosslinking reaction, and removing unreacted crosslinking agent and terminating the reducing agent by an ultrafiltration liquid exchange mode to obtain crosslinked hemoglobin;

wherein:

in the step a1, the concentration of the deoxyhemoglobin is diluted to 1-3g/dL, and a sulfhydryl compound is added into the hemoglobin, so that the mass ratio of the hemoglobin to the sulfhydryl compound in the solution is 5-10:1, wherein the sulfhydryl compound is N-acetyl-L-cysteine;

and the mass ratio of the hemoglobin to the cross-linking agent added into the reactor is 1000: 29-45.

In some embodiments, the crosslinking agent is glutaraldehyde.

In some embodiments, in step b1, the crosslinker is added at a rate of 50 to 500 mL/min.

In some embodiments, in step b1, the pressure of the reactor is between 1.05 and 1.25 bar.

In a fifth aspect of the invention, the invention provides an organ perfusion fluid comprising: cross-linked hemoglobin prepared by the foregoing method; vitamin K1; and prostacyclin.

In some embodiments, the organ perfusate comprises:

10-50g of cross-linked hemoglobin prepared by the above method

Human albumin (Shandongtai bang biology, pharmaceutical grade) 40-60g

Heparin (Chenxin pharmaceutical industry) 8000-12000IU

8.4% sodium bicarbonate (Tiandi pharmacy) 8-12mL

8-12mL of 10% calcium gluconate (Huarun double crane)

Vancomycin (North China pharmaceutical) 0.4-0.6mg

Gentamicin (North China pharmaceutical) 0.05-0.07mg

40-60mL of 10% compound amino acid injection (Huarui pharmaceutical)

Vitamin K1 (honest pharmaceutical industry) 0.05-0.15mg

2ug/mL prostacyclin (Mingren medicine) 8-32mL

In a sixth aspect of the present invention, there is provided a method for preparing cross-linked hemoglobin, comprising the steps of:

a2, the concentration of diluted deoxyhemoglobin is 1-3g/dL (such as 1.5g/dL, 2g/dL or 2.5 g/dL);

b2, adding glutaraldehyde as a cross-linking agent to the diluted deoxyhemoglobin at a ratio of 29-45mg (e.g., 31mg, 33mg, 35mg, 37mg, 39mg, 41mg or 43mg) of glutaraldehyde per 1g of hemoglobin to cause a cross-linking reaction;

c2, adding a reducing agent to stop the crosslinking reaction, and removing the unreacted crosslinking agent and the reducing agent by an ultrafiltration liquid exchange mode to obtain the crosslinked hemoglobin.

In some embodiments, the time for the crosslinking reaction is 40-60min (e.g., 45min, 50min, or 55 min).

In some embodiments, in step a2, a sulfhydryl compound is added to the diluted deoxyhemoglobin, and the mass ratio of hemoglobin to sulfhydryl compound is 5-10:1 (e.g., 6:1, 20:3, 7:1, 8:1, or 9: 1).

In some embodiments, the sulfhydryl compound is mercaptoethylamine, glutathione or N-acetyl-L-cysteine.

In some embodiments, in step b2, the crosslinker is added at a rate of 50-500mL/min (e.g., 100mL/min, 150mL/min, 200mL/min, 250mL/min, 260mL/min, 300mL/min, 350mL/min, 400mL/min, or 450 mL/min).

In some embodiments, the reducing agent is sodium borohydride.

In some embodiments, the ultrafiltration exchange fluid is conducted using a membrane module or hollow fiber column having a KD of 10KD to 100KD (e.g., 20KD, 30KD, 40KD, 50KD, 60KD, 70KD, 80KD, or 90 KD).

In a seventh aspect of the invention, there is provided an organ perfusion fluid comprising cross-linked hemoglobin prepared by the method described above.

In some embodiments, the organ perfusate further comprises vitamin K1 and prostacyclin.

In some embodiments, the organ perfusate comprises:

10-50g of cross-linked hemoglobin prepared by the above method

Human albumin (Shandongtai bang biology, pharmaceutical grade) 40-60g

Heparin (Chenxin pharmaceutical industry) 8000-12000IU

8.4% sodium bicarbonate (Tiandi pharmacy) 8-12mL

8-12mL of 10% calcium gluconate (Huarun double crane)

Vancomycin (North China pharmaceutical) 0.4-0.6mg

Gentamicin (North China pharmaceutical) 0.05-0.07mg

40-60mL of 10% compound amino acid injection (Huarui pharmaceutical)

Vitamin K1 (honest pharmaceutical industry) 0.05-0.15mg

2ug/mL prostacyclin (Mingren medicine) 8-32mL

In an eighth aspect of the present invention, the present invention provides a method for preparing the aforementioned organ perfusate, which comprises the following steps:

1) adding human albumin, heparin, 8.4% of sodium bicarbonate, 10% of calcium gluconate, vancomycin, gentamicin, 10% of compound amino acid injection, 12 compound vitamins for injection and vitamin K1 according to the formula proportion, quantifying distilled water, filtering for sterilization, aseptically filling and sealing to obtain a solution I;

2) deoxidizing the cross-linked hemoglobin prepared by the method, filtering and sterilizing, carrying out sterile and oxygen-free filling, and carrying out thermoplastic sealing to obtain a solution II;

3) providing separately packaged prostacyclin according to a formula proportion to obtain a solution III;

4) and uniformly mixing the solution I and the solution II according to a ratio, and performing infusion by matching with the solution III to obtain the organ perfusate.

Has the advantages that:

1. compared with the only available similar product oxyglobin and the polymerization method provided by patent 99808286.4, the polymerized hemoglobin prepared by the method has the polymer content lower by more than 10 percent, and meets the requirement of FDA on the molecular weight reduction of the product of biopure company;

2. the polymerization time of the invention can be completed within 1 hour, while the polymerization time of patent 99808286.4 is completed within about 5 hours, thereby greatly shortening the passing time, not only reducing the risks of pollution and excessive endotoxin, but also saving the production cost;

3. according to the preparation method of the cross-linked hemoglobin, NAC is added before cross-linking, so that the content of high molecular weight hemoglobin and low molecular weight hemoglobin is further reduced, the risk of nephrotoxicity and vasoconstriction caused by low molecular weight protein and the risk of blood viscosity increase, adverse blood flow and deposition on the inner wall of blood vessels, liver or spleen caused by high molecular weight protein are reduced;

4. the invention does not need complex equipment investment and expensive reagent purchase, has simple method and is easy to popularize and apply.

Drawings

FIG. 1 is a system connection schematic of an aggregation system according to some embodiments of the present disclosure;

FIG. 2 is a schematic diagram of system connections for an aggregation system in accordance with further embodiments of the present invention;

FIG. 3 is a schematic diagram showing the molecular weight distribution of crosslinked hemoglobin prepared by the method of example 13, wherein Dimer represents a Dimer, Tetramer represents a Tetramer, Octamer represents an Octamer, and Oligomer represents a polymer;

FIG. 4 is a schematic diagram showing the molecular weight distribution of crosslinked hemoglobin prepared by the method of example 14, wherein Dimer represents a Dimer, Tetramer represents a Tetramer, Octamer represents an Octamer, and Oligomer represents a polymer;

FIG. 5 is a schematic diagram showing the molecular weight distribution of crosslinked hemoglobin prepared by the method of example 15, wherein Dimer represents a Dimer, Tetramer represents a Tetramer, Octamer represents an Octamer, and Oligomer represents a polymer;

FIG. 6 is a schematic view of a product package for Oxyglobin;

FIG. 7 shows the results of measuring the molecular weight distribution of cross-linked hemoglobin prepared by polymerization according to the method of patent No. 99808286.4, wherein Dimer represents Dimer, Tetramer represents Tetramer, Octamer represents Octamer, and Oligomer represents macromer;

FIG. 8 shows the results of molecular weight distribution measurements of Oxyglobin, in which Dimer represents Dimer, Tetramer represents Tetramer, Octamer represents Octamer, and Oligomer represents polymer;

FIG. 9 is a pictorial view of a farm of cattle according to an embodiment of the present invention;

FIG. 10 is a live-action view of collected bovine blood according to an embodiment of the present invention;

FIG. 11 is a schematic flow chart of washing collected blood according to an embodiment of the present invention;

FIG. 12 is a schematic flow diagram of cell lysis and diafiltration according to an embodiment of the invention;

FIG. 13 is a schematic flow diagram of filtered deoxygenation according to an embodiment of the present invention;

FIG. 14 is a schematic flow diagram of chromatographic purification of hemoglobin according to an embodiment of the present invention;

FIG. 15A is a schematic flow chart illustrating deoxygenation of purified hemoglobin according to an embodiment of the present invention;

FIG. 15B is a schematic flow chart illustrating deoxygenation of purified hemoglobin according to an embodiment of the present invention;

FIG. 16 is a schematic flow chart of the polymerization of purified hemoglobin with glutaraldehyde according to an embodiment of the present invention;

FIG. 17 is a schematic diagram of a process for reducing glutaraldehyde-crosslinked hemoglobin with sodium borohydride in accordance with an embodiment of the present invention;

FIG. 18 is a schematic view of a gas residue-free canning apparatus according to an embodiment of the present invention;

FIG. 19 is a schematic view of a canned product according to an embodiment of the invention;

FIG. 20 is a schematic flow chart of washing the collected blood according to an embodiment of the present invention;

FIG. 21 is a schematic flow chart of cell lysis according to an embodiment of the present invention;

FIG. 22 is a schematic flow diagram of filtered deoxygenation according to an embodiment of the present invention;

FIG. 23 is a schematic flow diagram of chromatographic purification of hemoglobin according to an embodiment of the present invention;

FIG. 24A is a schematic flow chart illustrating deoxygenation of purified hemoglobin according to an embodiment of the present invention;

FIG. 24B is a schematic flow chart illustrating deoxygenation of purified hemoglobin according to an embodiment of the present invention;

FIG. 25 is a schematic flow chart of the polymerization of purified hemoglobin with glutaraldehyde according to an embodiment of the present invention;

FIG. 26 is a schematic diagram of a process for reducing glutaraldehyde-crosslinked hemoglobin with sodium borohydride in accordance with an embodiment of the present invention;

FIG. 27 is a schematic view of a sterile filtration process according to an embodiment of the present invention;

FIG. 28 is an image of a separation system used in the method of red blood cell purification according to an embodiment of the present invention;

FIG. 29 is a schematic view of a separation system used in the method for purifying erythrocytes according to the embodiment of the present invention;

FIG. 30 is an image of an aggregation assembly according to an embodiment of the present invention;

FIG. 31 is a tomographic field image of an embodiment of the present invention;

FIG. 32 is a graph showing the results of detection of column effect before sample application in accordance with the embodiment of the present invention;

FIG. 33 is a graph image of tomographic 1 data according to an embodiment of the present invention;

FIG. 34 is a graph image of tomographic 2 data according to an embodiment of the present invention;

FIG. 35 is an image of SDS-PAGE purity measurement of a collection obtained by chromatography 2 according to example of the present invention, wherein 1 denotes C500, 2 denotes AEX peak-crossing 1, 3 denotes AEX peak-crossing 2, 4 denotes AEX peak-washing, 5 denotes AEX peak-eluting, 6 denotes AEX peak-tailing, 7 denotes AEX peak-regenerating, and 8 denotes Marker;

FIG. 36 is an image of a primary filter arrangement according to an embodiment of the invention;

FIG. 37 is a schematic view of a primary filtration apparatus according to an embodiment of the present invention;

FIG. 38 is an image of a module for a 100KD diafiltration process according to an embodiment of the present invention;

FIG. 39 is a schematic diagram of components used in a 100KD diafiltration process according to an embodiment of the present invention;

FIG. 40 is an image of an assembly for a 30KD diafiltration concentration process according to an embodiment of the present invention;

FIG. 41 is an image of an assembly for degassing membrane deoxygenation treatment in accordance with an embodiment of the present invention;

FIG. 42 is a schematic view of an assembly for degassing membrane deoxygenation treatment in accordance with an embodiment of the present invention;

FIG. 43 is a design drawing for commercial scale production of an embodiment of the present invention;

FIG. 44 is a negative-level warehouse layout of an embodiment of the present invention;

FIG. 45 is a diagram of a two-level QC, development, utility room design according to an embodiment of the present invention;

fig. 46 is an EDQM certificate image obtained by a farm collecting bovine blood according to an embodiment of the present invention.

Reference numerals:

FIG. 1: 1-atomizer, 2-liquid inlet pipe, 3-air inlet pipe, 4-balance air pipeline, 5-liquid inlet pipe valve, 6-exhaust valve, 7-pressure gauge, 8-air inlet pipe valve, 9-atomization range, 10-liquid level, 11-bottom stirring rotor, 12-anaerobic reaction vessel and 13-water bath jacket;

FIG. 2: 1-static mixer, 2-circulating rubber tube, 3-liquid feeding rubber tube, 4-T type connector, 5-stopping clamp, 6-circulating hemoglobin flow direction, 7-ribbon, 8-magnetic stirrer, 9-polymerization reactor, 10-thermometer, 11-nitrogen circulating pipeline, 12-liquid feeding peristaltic pump, 13-circulating peristaltic pump and 14-diluted glutaraldehyde;

FIG. 18: the method comprises the following steps of 1-tank body, 2-inert gas source, 3-vacuum pump, 4-T-shaped pipeline, 41-tank body section, 42-gas source section, 43-vacuum section, 5-filling bag, 61-first cut-off switch, 62-second cut-off switch, 63-third cut-off switch, 64-flow stopping clamp, 7-peristaltic pump and 8-gas balance pipe.

Detailed Description

As used herein, unless otherwise indicated herein or otherwise evident from the context, the term "about" is to be understood as within the normal tolerance of the art, e.g., within 2 standard deviations of the mean. "about" can be understood as being within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value. Unless the context indicates otherwise, all numbers provided herein are to be modified by the term "about".

The phrase "aberrantly expressed" is used to refer to an expression level that deviates from (i.e., increases or decreases in expression level of) a normal reference expression level of a gene.

The term "agent" refers to any small protein or other compound, antibody, nucleic acid molecule or polypeptide, or fragment thereof.

"alteration" refers to a change (increase or decrease) in the molecular weight distribution of a stabilization technique or reaction, as detected by standard art-known methods such as those described herein. As used herein, a change includes at least a 5% change in the level of crosslinking, for example at least a 5% to 95% or 100% change in the level of stabilization of the crosslinking molecule. For example, the alteration comprises at least a 5% -10% change in protein stability, preferably a 25% change, more preferably an 80% change, and most preferably a 590% or greater change in the size of the stabilizing molecule.

"improving" refers to reducing, inhibiting, attenuating, reducing, arresting or stabilizing the development or progression of a disease.

The term "antibody" (Ab) as used herein includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity. The term "immunoglobulin" (Ig) is used interchangeably herein with "antibody".

By "binding" a molecule is meant having a physicochemical affinity for the molecule.

"control" or "reference" refers to a comparative standard. In one aspect, as used herein, a sample or subject that is "altered as compared to a control" is understood to have a level that is statistically different from a normal, untreated or control sample. Control samples include, for example, cells in culture, one or more laboratory test animals, or one or more human subjects. Methods of selecting and testing control samples are within the ability of those skilled in the art. The analyte may be a naturally occurring substance that is expressed or produced by a cell or organism (e.g., an antibody, a protein), or a substance that is produced by a reactive substance to form a covalent bond (e.g., glutaraldehyde). The amount of change and the measured value may vary depending on the method used for detection. The determination of statistical significance is within the ability of those skilled in the art, e.g., the number of standard deviations from the mean that constitute a positive result.

"detecting" refers to identifying the presence, absence, or amount of an agent to be detected, e.g., a nucleic acid molecule, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

"detectable label" refers to a composition that, when attached (e.g., directly or indirectly) to a molecule of interest, allows the latter to be detected, for example, by spectroscopic, photochemical, biochemical, immunochemical, or other chemical means. Direct labeling may occur through a bond or interaction linking the label to the molecule, while indirect labeling may occur through the use of a linker or bridging moiety that is directly or indirectly labeled.

The "detecting step" can use any of a variety of known methods to detect the presence of a nucleic acid (e.g., methylated DNA) or polypeptide. Types of detection methods in which probes may be used include Western blots, Southern blots, dot or slot blots and Northern blots.

The terms "effective amount" and "therapeutically effective amount" of a formulation or formulation component refer to a sufficient amount of the formulation or component, alone or in combination, to provide the desired effect. For example, an "effective amount F" refers to the amount of a compound, alone or in combination, required to ameliorate the symptoms of anemia and/or iron deficiency relative to an untreated patient. The effective amount of active compound for use in the practice of the present invention for the therapeutic treatment of a disease will depend on the mode of administration, the age, weight and general health of the subject. Finally, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. Such an amount is referred to as an "effective" amount.

The term "fragment" refers to a portion of a protein molecule. This portion preferably comprises at least the molecule of hemoglobin or the heme iron portion of the original protein construct. For example, the fragment may comprise 1, 2 or 4 side chains of the native hemoglobin molecule and of the beta fragment. However, the present invention also includes such protein fragments so long as they exhibit the desired biological activity from the full-length globular protein structure, e.g., in many embodiments of the invention including illustrative polyamino acid fragments of total weight of about 16000kD, about 32000kD (including all intermediate weights). Similarly, if the protein fragment is a siderophore (heme group), protein fragments of almost any length are used.

The terms "isolated," "purified," or "biologically pure" refer to a material that is not associated to a varying degree with the components that normally accompany it as found in its natural environment. "isolated" refers to the degree of separation from the original source or environment. "purified" means separated by a higher degree of separation.

A "purified" or "biologically pure" protein is sufficiently free of other materials that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, the stable protein of the polymer fragment of the present invention is purified if it is substantially free of cellular material, viral material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized, as well as all other stromal red blood cells or other blood proteins or blood components and cellular debris. Purity, homogeneity and stability are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" may mean that the nucleic acid or protein produces a substantial band in the electrophoresis gel. For proteins that can be modified, e.g., phosphorylated, glycosylated or polymerized, different modifications can result in different isolated proteins that can be purified separately.

Similarly, "substantially pure" refers to a protein or polypeptide that is isolated from the components that naturally accompany it. Typically, proteins and polypeptides are substantially pure when they are present in at least 95% and even 99% of their content, free of other proteins and naturally occurring organic molecules with which they are naturally associated.

An "isolated polypeptide" refers to a polypeptide of the invention that is isolated from components that naturally accompany it. Typically, a polypeptide is isolated free of proteins and naturally occurring organic molecules with which it is naturally associated when the polypeptide is at least 60% by weight. Preferably, the preparation is at least 75% in content, more preferably at least 90% in content, most preferably at least 99% in content of the polypeptide of the invention. The isolated polypeptide fraction and/or protein of the invention may be obtained, for example, by extraction from a natural source, by expression of recombinant nucleic acids encoding such a substance; or by chemical synthesis of the protein. Purity can be measured by any suitable method, such as column chromatography, polyacrylamide gel, electrophoresis or HPLC analysis.

The term "immobilized" or "attached" refers to a probe (e.g., a nucleic acid or protein) and a solid support, wherein the binding between the probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal. The binding may be covalent or non-covalent. The covalent bond may be formed directly between the probe and the solid support, or may be formed by a cross-linking agent, or by the inclusion of a specific reactive group on the solid support or the probe or both molecules. The non-covalent binding may be one or more of electrostatic, hydrophilic and hydrophobic interactions. Non-covalent binding includes covalent attachment of the molecule to a carrier and non-covalent binding of the biotinylated probe to the molecule. Immobilization may also involve a combination of covalent and non-covalent interactions.

The term "marker" refers to any protein or polynucleotide having an altered expression level or activity associated with a disease or disorder, such as neoplasia.

"modulate" refers to alteration (increase or decrease). These changes are detected by standard art-known methods, such as those described herein.

The term "normal amount" refers to a normal amount of the complex in an individual who is not known to be diagnosed with cancer or various metabolic and physiological disease states. The amount of molecules can be measured in a test sample and compared to a "normal control level" using techniques such as reference limits, discrimination limits or risk definition thresholds to define cut-off points and outliers (e.g., for neoplasia, hypoxia, ischemia). "normal control level" refers to the level of one or more proteins (or nucleic acids) or a combined protein index (or combined nucleic acid index) typically found in a subject known not to have cancer or a physiological hypoxic state. Such normal control levels and cut-offs can vary depending on whether the molecule is used alone or in a formula that combines other proteins into an index. Alternatively, the normal control level may be a database of protein patterns from previously tested subjects that have not converted to cancer within a clinically relevant time period. It may also be a condition of reduced oxygen tension, measured as MMHG, characterized by hypoxia or ischemia. In another aspect, the normal control level can be a level relative to normal cellular function and oxidation level.

The determined level may be the same as the control level or the cutoff level or the threshold level, or may be increased or decreased with respect to the control level or the cutoff level or the threshold level. In some aspects, the control subject is a matched control of the same species, gender, ethnicity, age group, smoking status, Body Mass Index (BMI), current treatment regimen status, medical history, or a combination thereof, but unlike the subject diagnosed and evaluated, the control does not suffer from the disease or is not at risk of suffering from the disease or reflects signs and symptoms of hypoxia.

The determined level may be an increased level relative to the control level. As used herein, the term "increased relative to a level (e.g., expression level, biological activity level, etc.)" refers to any% increase over a control level. The level of increase may be at least or about a 5% increase, at least or about a 10% increase, at least or about a 15% increase, at least or about a 20% increase, at least or about a 25% increase, at least or about a 30% increase, at least or about a 35% increase, at least or about a 40% increase, at least or about a 45% increase, at least or about a 50% increase, at least or about a 55% increase, at least or about a 60% increase, at least or about a 65% increase, at least or about a 70% increase, at least or about a 75% increase, at least or about an 80% increase, at least or about an 85% increase, at least or about a 90% increase, or at least or about a 95% increase, relative.

The determined level may be a reduced level relative to the control level. As used herein, the term "reduced relative to a level (e.g., expression level, biological activity level, etc.)" refers to any% reduction below a control level. The level of reduction may be at least or about a 1% reduction, at least or about a 5% reduction, at least or about a 10% reduction, at least or about a 15% reduction, at least or about a 20% reduction, at least or about a 25% reduction, at least or about a 30% reduction, at least or about a 35% reduction, at least or about a 40% reduction, at least or about a 45% reduction, at least or about a 50% reduction, at least or about a 55% reduction, at least or about a 60% reduction, at least or about a 65% reduction, at least or about a 70% reduction, at least or about a 75% reduction, at least or about an 80% reduction, at least or about an 85% reduction, at least or about a 90% reduction, or at least or about a 95.

Protein molecules useful in the methods of the invention include any nucleic acid molecule encoding a polypeptide of a heme iron composition of the invention, or a fragment thereof. Such protein-stable molecules need not be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity, e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity.

For most applications, the stringency of the washing steps after hybridization will also vary. The strictly controlled washing/mixing conditions can be defined by the buffer concentration, the dispersion conditions and the temperature of the glutaraldehyde reaction. As described above, the controlled stringency can be increased by reducing the salt concentration or by increasing the temperature. Other variations of these conditions will be apparent to those skilled in the art. Hybridization/conjugation techniques are well known to those skilled in the art and are described, for example, in Benton and Advis (Science196:180, 1977); grunstein and Hogness (proc.natl.acad.sci., USA 72:3961, 1975); ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); berger and Kimmel (targeting molecular cloning technology, 1987, Academic Press, New York); and Sambrook et al, Molecular Cloning, A Laboratory Manual, Cold Spring harbor Laboratory Press, New York.

"neoplasia" refers to a disease or disorder characterized by hyperproliferation or reduced apoptosis. Illustrative tumors in which the invention may be used include, but are not limited to, pancreatic cancer, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythrocytic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphomas (hodgkin's disease, non-hodgkin's disease), wald's macroglobulinemia, heavy chain diseases and solid tumors such as sarcomas and carcinomas (e.g., malignant lymphoma), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyoma, rhabdomyosarcoma, colon cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, bladder cancer, intramedullary cancer, bronchial cancer, renal cell carcinoma, liver cancer, nile ductal carcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, epididymoma, spongioblastoma, acoustic neuroma, oligodendroglioma, glioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

As used herein, "obtaining" as described in "obtaining an agent" includes synthesizing, purchasing, or otherwise obtaining an agent.

As used herein, the term "or" is to be understood as being inclusive unless specifically indicated or otherwise evident from the context. As used herein, the terms "a", "an" and "the" are to be construed as either singular or plural unless otherwise indicated herein or apparent from the context.

The phrase "pharmaceutically acceptable carrier" is well known in the art and includes pharmaceutically acceptable materials, compositions or excipients suitable for administration of the compounds of the present invention to a mammal. The carrier comprises a liquid or solid filler, diluent, excipient, solvent or encapsulating material involved in carrying or transporting the subject agent from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include sugars such as lactose, glucose and sucrose; gelatin; an excipient; pyrogen-free water; isotonic saline; ringer's solution; ethanol; phosphate buffer; and other non-toxic compatible materials used in pharmaceutical formulations.

The term "protein" or "polypeptide" or "peptide" refers to any chain of more than two natural or unnatural amino acids, regardless of post-translational modifications (e.g., glycosylation or phosphorylation), as described herein, making up all or part of a naturally occurring or non-naturally occurring polypeptide or peptide.

A "primer set" refers to a set of oligonucleotides that can be used, for example, in PCR. The primer set consists of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 80,100,200,250,300,400,500,600 or more primers.

The term "prevention" refers to the administration of an agent or composition to a clinically asymptomatic individual at risk of developing, susceptible to or susceptible to a particular adverse condition, disorder or disease, and thus relates to the prevention of the occurrence of symptoms and/or their underlying cause.

Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It will also be understood that a plurality of values are disclosed herein, and that each value is also disclosed herein as "about" that particular value, in addition to the value itself. It should also be understood that throughout the application, data is provided in a number of different formats and represents endpoints, starting points, and ranges for any combination of data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that greater than, greater than or equal to, less than or equal to, and equal to 10 and 15 and between 10 and 15 are considered disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13 and 14 are also disclosed.

Ranges provided herein are to be understood as shorthand for all values falling within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange selected from 1 to 50, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 and all intervening fractional values between the foregoing integers, e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, "nested subranges" extending from either end of the range are specifically contemplated. For example, nested sub-ranges of exemplary ranges 1-50 may include 1-10, 1-20, 1-30, and 1-40 in one direction, or 50-40, 50-30, 50-20, and 50-10 in another direction.

By "reduced" is meant a negative change of at least 10%, 25%, 50%, 75% or 100%.

A "reference sequence" is a defined sequence that is used as a basis for sequence comparison or comparison of gene expression. The reference sequence may be a subset or all of the specified sequence; for example, a fragment of a full-length cDNA or gene sequence, or the entire cDNA or gene sequence. For polypeptides, the length of a reference polypeptide sequence is typically at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, even more preferably about 35 amino acids, about 50 amino acids or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence is typically at least about 40 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, even more preferably about 100 nucleotides or about 300 or about 500 nucleotides or any integer therebetween.

The term "sample" as used herein refers to a biological sample obtained for in vitro evaluation. Exemplary tissue samples for the methods described herein include tissue samples from neoplasia or circulating exosomes. With respect to the methods disclosed herein, the sample or patient sample preferably may comprise any body fluid or tissue. In some embodiments, the bodily fluid includes, but is not limited to, blood, plasma, serum, lymph, breast milk, saliva, mucus, semen, vaginal secretions, cellular extracts, inflammatory fluids, cerebrospinal fluid, feces, vitreous humor, or urine obtained from the subject. In some aspects, the sample is a composite panel of at least two of a blood sample, a plasma sample, a serum sample, and a urine sample. In exemplary aspects, the sample comprises blood or a fraction thereof (e.g., plasma, serum, a fraction obtained by leukocyte isolation). Preferred samples are whole blood, serum, plasma or urine. The sample may also be a partially purified fraction of a tissue or body fluid.

The reference sample may be a "normal" sample, from a donor without the disease or condition fluid, or from normal tissue of a subject with the disease or condition. The reference sample can also be from an untreated donor or cell culture that is not treated with the active agent (e.g., no treatment or administration of the carrier alone). A reference sample may also be taken at a "zero time point" prior to contacting the cell or subject with the agent to be tested or therapeutic intervention or at the start of the intended study.

"solid support" describes a strip, polymer, bead or nanoparticle. The strip may be a nucleic acid probe (or protein) -coated porous or non-porous solid support strip comprising a nucleic acid probe linked to a support to prepare a conjugate and immobilizing the conjugate on a porous solid support. Well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, Gabbros and magnetite. For the purposes of the present invention, the nature of the carrier may be soluble or insoluble to some extent. The support material may have virtually any possible structural configuration, as long as the conjugated molecule is capable of binding to a binding agent (e.g., an antibody or nucleic acid molecule). Thus, the support structure may be spherical, such as beads, or cylindrical, such as the inner surface of a test tube, or the outer surface of a rod. Alternatively, the surface may be flat, such as a sheet, or a test strip, etc. For example, the support comprises polystyrene beads. Those skilled in the art will know of many other suitable carriers for binding antibodies or antigens, or be able to determine such carriers by routine experimentation. In other aspects, the solid support comprises a polymer to which the agent is chemically bound, immobilized, dispersed, or associated. The polymeric support may be a polymeric network and may be prepared in bead form (e.g. by suspension polymerisation). The location of the active site incorporated into the polymeric carrier depends on the type of polymeric carrier. For example, in a swollen gel bead polymer support, the active sites are uniformly distributed throughout the bead, whereas in a macroporous bead polymer support, they are predominantly on the inner surface of the macropores. The solid support, e.g. device, comprises a binding agent, either alone or together with a binding agent for at least one, two, three or more other molecules.

The term "specifically binds" refers to a compound or antibody that recognizes and binds to a polypeptide of the invention, but which does not substantially recognize and bind to other molecules in a sample, such as a biological sample, which naturally includes a polypeptide/conjugated purified protein of the invention.

"substantially identical" refers to a polypeptide/protein or nucleic acid molecule (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein) that is at least 80% identical to a reference amino acid sequence. Preferably, such sequences are at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

The term "subject" as used herein includes all members of the animal family susceptible to the disorder. In some aspects, the subject is a mammal, and in some aspects, the subject is a human. The method is also applicable to companion animals such as dogs and cats as well as livestock such as cows, horses, sheep, goats, pigs and other domesticated and wild animals.

A subject, disorder or syndrome "having or suspected of having" a particular disease has a sufficient number of risk factors, or exhibits a sufficient number of signs or symptoms of the disease, disorder or syndrome, or a combination of these signs or symptoms, such that a capable individual will diagnose or suspect that the subject has the disease, disorder or syndrome. Methods for identifying subjects having or suspected of having a cancer-associated disorder are within the ability of those skilled in the art. The subjects having and suspected of having a particular disease, disorder or syndrome need not be in two distinct groups.

As used herein, "susceptible to" or "at risk of developing a particular disease or disorder" refers to an individual that is more likely to develop a disease or disorder than the general population based on genetic, environmental, health, and/or other risk factors. The increase in likelihood of developing a disease may be an increase of about 10%, 20%, 50%, 100%, 150%, 200% or more.

The term "treating" as used herein refers to administering an agent or formulation to a clinically symptomatic individual with an adverse condition, thereby reducing the severity and/or frequency of the symptoms, eliminating the symptoms and/or their underlying cause, and/or promoting amelioration or repair of the injury. It will be appreciated that although treatment of a disease or condition is not precluded, it is not necessary to completely eliminate the disease, condition or symptom associated therewith.

In some cases, the compositions of the present invention are administered orally or systemically. Other modes of administration include topical, intraocular, buccal, intra/over implant or parenteral routes. The term "parenteral" includes subcutaneous, intrathecal, intravenous, intramuscular, intraperitoneal or infusion. The intravenous or intramuscular routes are not particularly suitable for long-term treatment and prophylaxis. However, in emergency situations, they may be preferred. Compositions comprising the compositions of the present invention may be added to physiological fluids such as blood. Oral administration may be preferred for prophylactic treatment due to patient convenience and dosing schedules. Parenteral administration (subcutaneous or intravenous) is preferred for more acute conditions or for the treatment of patients who cannot tolerate enteral administration due to gastrointestinal intolerance, ileus or other concomitant critical conditions.

The pharmaceutical composition may be assembled into a kit or a pharmaceutical system for use in the adjuvant treatment of the cell cycle of rapidly dividing cells, such as cancer cells. The kit or pharmaceutical system according to this aspect of the invention comprises a carrier means, such as a box, carton, tube, having one or more container means therein, such as a vial, tube, ampoule, bottle, syringe or bag, said container means being tightly confined therein. The kits or pharmaceutical systems of the invention may also include instructions for use of the kit.

Any of the compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

The transitional term "comprising" which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional unrecited elements or method steps. Rather, the transitional phrase "consisting essentially of" limiting the scope of the claims to the specified elements or components "includes excluding any elements, steps, or components not specified in the claims. The transitional phrase "consisting essentially of the material or steps that limit the scope of the claims to the specified material or steps" and those that do not materially affect the basic and novel characteristics of the claimed invention.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The invention aims to solve the technical problem of providing a preparation method of cross-linked hemoglobin, the cross-linked hemoglobin prepared by the method contains low-content macromolecules (more than 500kD) and small molecules (less than 64kD), the cross-linked hemoglobin is added into an organ perfusate, the problems of organ acidosis, poor activation of lysosomal enzyme and cell damage caused by excessive free radicals generated during reperfusion due to lack of an oxygen carrying agent in the existing perfusate can be solved, and the preparation, storage, transportation and use of the cross-linked hemoglobin are very simple and convenient.

In order to solve the above problems, the present invention further provides a polymerization process for obtaining a low high polymer (molecular weight. gtoreq.192 kDa) content, which is more than 10% lower than the high polymer content of glutaraldehyde-polymerized hemoglobin provided in patent 99808286.4 and oxyglobin as the only commercially available product.

The invention provides a method for obtaining glutaraldehyde polymerized hemoglobin with low high polymer (molecular weight is more than or equal to 192kDa), which is mainly an improvement on the basis of the method of patent 99808286.4, and the method gradually searches key factors influencing the molecular weight distribution of polymerization through multiple polymerization tests and obtains a suitable parameter range through further tests on the factors. The method comprises the following steps: before polymerization, washing and assembling a polymerization system, diluting and heating a hemoglobin solution, adding a diluted cross-linking agent glutaraldehyde according to a proportion, adjusting the pH value, adding sodium borohydride to terminate the polymerization reaction, and performing ultrafiltration with 30KD to remove residual glutaraldehyde and sodium borohydride to obtain polymerized hemoglobin. The specific process of polymerization is as follows: washing pipelines, containers and accessories with 0.5N sodium hydroxide solution before polymerization to remove pyrogens, washing with injection water to neutrality, assembling a polymerization system according to a schematic diagram 2, particularly paying attention to the assembly of static mixer components, inserting a static mixer into a peristaltic pump pipe, bundling the tail end of a liquid flow direction with a binding belt to prevent the static mixer from moving away when the liquid flows, connecting branch pipes added with glutaraldehyde, pH adjusting buffer solution and sodium borohydride buffer solution through a T-shaped connector, mounting flow clamps on the branch pipes to prevent backflow of the liquid, and after the assembly is completed, carrying out autoclaving at 121 ℃ for 30 min. Assembling a polymerization system according to a schematic diagram 2, blowing nitrogen through a nitrogen circulating pipeline and keeping the nitrogen till the polymerization is finished, calculating the amount of hemoglobin to be added, measuring and adding the amount of the hemoglobin into a polymerization reaction kettle, diluting the hemoglobin into 1-3g/dL by ultrapure water (injection water), adding NAC according to the proportion of 2mg/mL of final concentration, heating the diluted hemoglobin solution to 42 +/-2 ℃ by a heating sleeve of the reaction kettle, taking the temperature according to the value of a reading thermometer, measuring glutaraldehyde according to the proportion of 29-45mg added to 1g of hemoglobin, diluting the glutaraldehyde into 6.3mg/mL by the ultrapure water (injection water), starting a peristaltic pump to circulate the hemoglobin and a magnetic stirrer to uniformly mix the hemoglobin, adding the diluted glutaraldehyde through a peristaltic pump within 50 minutes by a T-shaped connector, preserving heat for 20 minutes after the polymerization is finished, and then reducing the temperature to 25-20 ℃, adjusting the pH of the hemoglobin polymerization solution to 10 +/-0.2 by using a pH12.5 sodium borate buffer solution (38 g/L of borax), weighing sodium borohydride according to the proportion of 15.6g of 1g of hemoglobin, diluting the sodium borohydride to 9.5mg/mL by using a pH10.5 sodium borate buffer solution (4.6 g/L of borax), adding the sodium borohydride buffer solution within 15min through a T-shaped connector, changing the solution to the pH7.8 of the polymerization solution by using a 30KD ultrafiltration membrane pH7.6 disodium hydrogen phosphate solution changing buffer solution, and then changing the solution to a pH7.6 dialysis buffer solution C (6.7g/L of sodium chloride, 0.3g/L of potassium chloride, 0.15g/L of calcium chloride, 2.0 g/LN-acetylcysteine and 3.1g/L of sodium lactate) to obtain the polymerized hemoglobin.

The invention provides two detection methods of polymerized hemoglobin molecular weight distribution:

the detection method comprises the following steps:

1.1 reagents and materials used

1.2 instruments and installations

1.3 detection method

1.3.1 sample preparation

1.3.1.1 transfer 600. mu.L of the test sample to a 1.5mL centrifuge tube;

centrifuging at 13000rpm for 10min at 1.3.1.24 deg.C;

1.3.1.3 transfer supernatant to HPLC bottles.

1.4 detection method

1.4.1 chromatographyColumn: agilent advanced biosec2.7um,

7.8×300mm；

1.4.2 parameter settings

Parameter(s)	Is provided with
		Run time	30min
Flow rate of flow	0.5mL/min
		Column temperature
	30℃
		Temperature of the sample	10℃
Procedure of equal proportions	100% mobile phase
		VWD detector	Chromatogram extracted at 280nm

1.4.3 the polymerized hemoglobin is loaded at 20 μ g per needle for detection and the results are recorded.

And a second detection method comprises the following steps:

2.1 detection of the main materials to be prepared:

2.2 detection of the main devices to be prepared:

2.3 preparation of buffers required for detection:

buffer 750mM MgCl was prepared as follows₂，50mM tris，0.1mM EDTA pH 6.5。

2.3.1A 1000mL measuring cylinder was used to measure 800mL of ultrapure water and transferred to a 1L beaker.

2.3.2 to a 1L beaker, 10.46. + -. 0.05g of Bis-Tris was added and gently stirred with a magnetic stir bar until complete dissolution.

2.3.3 the pH of the solution was adjusted to 6.7. + -. 0.1 with concentrated HCl and gently stirred.

2.3.4 to a 1L beaker 152.48. + -. 0.05g of MgCl was added₂·6H2O。

2.3.5 to a 1L beaker was added 0.030. + -. 0.001g of EDTA and gently stirred with a magnetic stir bar until complete dissolution.

2.3.6 the pH of the solution was adjusted to 6.5. + -. 0.1 with concentrated hydrochloric acid and gently stirred.

2.3.7 adjust the pH to the desired range, use a 1000ml graduated cylinder and increase the volume to 1000ml with ultra pure water.

2.3.8 the solution was filtered using a 0.1 μm PES filter.

2.4 preparation of samples

2.4.1 Standard preparation (Bio Rad Gel Filtration Standard)

2.4.1.1 Add 500. mu.L of ultrapure water to the standard and spin gently.

2.4.1.2 were allowed to stand on ice for 2-3 minutes and the solution was transferred to a 1.5ml EP tube.

2.4.1.313000 rpm, and centrifuging at 4 ℃ for 10 minutes.

2.4.1.4 transfer the supernatant to a high performance liquid chromatography vial.

2.4.2 preparation of test samples

2.4.2.1 the diluted sample was transferred to a 1.5ml EP tube.

2.4.2.213000 rpm, and centrifuging at 4 ℃ for 10 minutes.

2.4.2.3 transfer the supernatant to a high performance liquid chromatography vial.

2.5 detection method

2.5.1 chromatography column used: agilent advanced Bio SEC2.7 μm,

7.8×300mm

2.5.2 parameter settings

Parameter(s)	Is provided with
		Run time	30min
Flow rate of flow	0.5mL/min
		Column temperature	25℃
Temperature of the sample	25℃
		Basic operation instruction program	100% mobile phase
VWD detector	Absorption at 280nm
		MALLS (Multi-angle laser scattering instrument)	DAWN EOS,λ＝690nm

2.5.3 the test data is saved and analyzed.

The second detection method is a method for analyzing the molecular weight distribution described in section [ 0041 ] of the specification of priority document 201910846580.9, which is described in further detail, and does not substantially change, and therefore does not affect the detection result.

The invention is further illustrated by the following examples.

Preparation of crosslinked hemoglobin and influence of different factors on molecular weight distribution of crosslinked hemoglobin

It should be noted that, when the polymerized hemoglobin product is measured by the molecular weight distribution measurement method one described above, the data of each group (dimer content, tetramer plus octamer content, and high polymer content) are rounded off, and therefore, the result of adding up to 100% may be caused.

Example 1 preparation of crosslinked hemoglobin

As shown in figure 1, diluting deoxygenated hemoglobin to 2g/dL, adding NAC with final concentration of 3mg/mL, moving it into an anaerobic reaction vessel 12, the liquid level 10 of which does not exceed half of the height of the anaerobic reaction vessel 12, fixing a pressure atomizer 1 on the top of the anaerobic reaction vessel 12, connecting an air inlet pipe 3 and an liquid inlet pipe 2 on the atomizer 1, connecting a balance air pipeline 4 on the top of the anaerobic reaction vessel 12 to adjust the required pressure in the anaerobic reaction vessel 12, closing the anaerobic reaction vessel 12, starting a bottom stirring rotor 11, heating the reaction liquid to 42 ℃ through a water bath jacket 13, opening a liquid inlet pipe valve 5, adding a cross-linking agent glutaraldehyde, adjusting the liquid inlet speed to 260mL/min, opening an air inlet pipe valve 8, introducing an inert gas, preferably high-purity nitrogen, adjusting the gas flow rate to ensure that the cross-linking agent mist particles are not sprayed on the wall of the anaerobic reaction vessel 12, and contact with the reaction liquid level 10 in the largest area, and (3) regulating and controlling an exhaust valve 6 on the balance gas pipeline 4 to balance the pressure of the oxygen-free reaction vessel 12 to 1.15bar, adding a crosslinking agent glutaraldehyde according to the proportion of 1g of hemoglobin to 37mg of glutaraldehyde until the end, stopping sodium borohydride, and purifying a 30kD ultrafiltration exchange solution to obtain the crosslinked hemoglobin.

The polymerized hemoglobin product of this example was tested using the first molecular weight distribution test method, and the results were: dimer content 4.02%, tetramer plus octamer content 69.60%, high polymer content 26.38%.

The polymerized hemoglobin product of this example was tested using the second molecular weight distribution test method, and the results were: the content of MW >500Kd (< 10%) is 4.3%, and the content of MW <64kD (< 5%) is 3.4%.

EXAMPLE 2 preparation of crosslinked hemoglobin

As shown in figure 1, diluting deoxygenated hemoglobin to 3g/dL, adding NAC with final concentration of 3mg/mL, moving it into an anaerobic reaction vessel 12, the liquid level 10 of which does not exceed half of the height of the anaerobic reaction vessel 12, fixing a pressure atomizer 1 on the top of the anaerobic reaction vessel 12, connecting an air inlet pipe 3 and an liquid inlet pipe 2 on the atomizer 1, connecting a balance air pipeline 4 on the top of the anaerobic reaction vessel 12 to adjust the required pressure in the anaerobic reaction vessel 12, closing the anaerobic reaction vessel 12, starting a bottom stirring rotor 11, heating the reaction liquid to 42 ℃ through a water bath jacket 13, opening a liquid inlet pipe valve 5, adding a cross-linking agent glutaraldehyde, adjusting the liquid inlet speed to 500mL/min, opening an air inlet pipe valve 8, introducing an inert gas, preferably high-purity nitrogen, adjusting the gas flow rate to ensure that the cross-linking agent mist particles are not sprayed on the wall of the anaerobic reaction vessel 12, and contact with the reaction liquid level 10 in the largest area, and adjusting and controlling an exhaust valve 6 on the balance gas pipeline 4 to balance the pressure of the oxygen-free reaction vessel 12 to 1.25bar, adding a crosslinking agent glutaraldehyde according to the proportion of 1g of hemoglobin to 45mg of glutaraldehyde until the addition is finished, stopping sodium borohydride, and purifying a 30kD ultrafiltration exchange solution to obtain the crosslinked hemoglobin.

The polymerized hemoglobin product of this example was tested using the first molecular weight distribution test method, and the results were: dimer content 3.43%, tetramer plus octamer content 58.98%, high polymer content 37.59%.

The polymerized hemoglobin product of this example was tested using the second molecular weight distribution test method, and the results were: the MW >500Kd (< 10%) content is 6.5%, the MW <64kD (< 5%) content is 2.4%.

EXAMPLE 3 preparation of crosslinked hemoglobin

As shown in figure 1, diluting deoxygenated hemoglobin to 1g/dL, adding NAC with a final concentration of 1mg/mL, moving it into an anaerobic reaction vessel 12, the liquid level 10 of which does not exceed half of the height of the anaerobic reaction vessel 12, fixing a pressure atomizer 1 on the top of the anaerobic reaction vessel 12, connecting an air inlet pipe 3 and an liquid inlet pipe 2 on the atomizer 1, connecting a balance air pipeline 4 on the top of the anaerobic reaction vessel 12 to adjust the required pressure in the anaerobic reaction vessel 12, closing the anaerobic reaction vessel 12, starting a bottom stirring rotor 11, heating the reaction liquid to 40 ℃ through a water bath jacket 13, opening a liquid inlet pipe valve 5, adding a cross-linking agent glutaraldehyde, adjusting the liquid inlet speed to 50mL/min, opening an air inlet pipe valve 8, introducing an inert gas, preferably high-purity nitrogen, adjusting the gas flow rate to ensure that the cross-linking agent mist particles are not sprayed on the wall of the anaerobic reaction vessel 12, and contact with the reaction liquid level 10 in the largest, and adjusting and controlling an exhaust valve 6 on the balance gas pipeline 4 to balance the pressure of the oxygen-free reaction vessel 12 to 1.05bar, adding a cross-linking agent glutaraldehyde according to the proportion of 1g of hemoglobin to 29mg of glutaraldehyde until the end, stopping sodium borohydride, and purifying a 30kD ultrafiltration exchange solution to obtain the cross-linked hemoglobin.

The polymerized hemoglobin product of this example was tested using the first molecular weight distribution test method, and the results were: dimer content 4.83%, tetramer plus octamer content 68.45%, high polymer content 26.72%;

the polymerized hemoglobin product of this example was tested using the second molecular weight distribution test method, and the results were: the MW >500Kd (< 10%) content is 2.7%, the MW <64kD (< 5%) content is 4.8%.

EXAMPLE 4 preparation of crosslinked hemoglobin

The same preparation method as in example 1, except that the concentration of NAC in the solution was 2mg/mL, i.e., the ratio of the concentration of hemoglobin to NAC was 10: 1.

the polymerized hemoglobin product of this example was tested using the second molecular weight distribution test method, and the results were: the MW >500Kd (< 10%) content is 3.9%, the MW <64Kd (< 5%) content is 4.1%.

EXAMPLE 5 preparation of crosslinked hemoglobin

The same preparation method as in example 1, except that the concentration of NAC in the solution was 4mg/mL, i.e., the ratio of the concentration of hemoglobin to NAC was 5: 1.

the polymerized hemoglobin product of this example was tested using the second molecular weight distribution test method, and the results were: the content of MW >500Kd (< 10%) is 4.2%, and the content of MW <64kD (< 5%) is 3.3%.

EXAMPLE 6 preparation of crosslinked hemoglobin

The same procedure as in example 1 was conducted except that the pressure in the reactor was atmospheric pressure.

The polymerized hemoglobin product of this example was tested using the second molecular weight distribution test method, and the results were: the MW >500Kd (< 10%) content is 6.7%, the MW <64Kd (< 5%) content is 4.5%.

Example 7 preparation of crosslinked hemoglobin

The same procedure as in example 1 was followed, except that the pressure in the reactor was 1.05 bar.

The polymerized hemoglobin product of this example was tested using the second molecular weight distribution test method, and the results were: the MW >500Kd (< 10%) content is 5.6%, the MW <64Kd (< 5%) content is 3.8%.

EXAMPLE 8 preparation of crosslinked hemoglobin

The same procedure as in example 1 was followed, except that the pressure in the reactor was 1.25 bar.

The polymerized hemoglobin product of this example was tested using the second molecular weight distribution test method, and the results were: the content of MW >500Kd (< 10%) is 4.2%, and the content of MW <64kD (< 5%) is 3.4%.

EXAMPLE 9 preparation of crosslinked hemoglobin

The same procedure as in example 1 was followed, except that glutaraldehyde, a crosslinking agent, was added at a rate of 500 mL/min.

The polymerized hemoglobin product of this example was tested using the second molecular weight distribution test method, and the results were: the MW >500Kd (< 10%) content is 3.6%, the MW <64Kd (< 5%) content is 4.7%.

EXAMPLE 10 preparation of crosslinked hemoglobin

The same procedure as in example 1 was followed, except that glutaraldehyde, a crosslinking agent, was added at a rate of 200 mL/min.

The polymerized hemoglobin product of this example was tested using the second molecular weight distribution test method, and the results were: the content of MW >500Kd (< 10%) is 4.3%, and the content of MW <64kD (< 5%) is 3.3%.

EXAMPLE 11 preparation of crosslinked hemoglobin

The same procedure as in example 1 was followed, except that glutaraldehyde, a crosslinking agent, was added at a rate of 50 mL/min.

The polymerized hemoglobin product of this example was tested using the second molecular weight distribution test method, and the results were: the content of MW >500Kd (< 10%) is 4.7%, and the content of MW <64kD (< 5%) is 2.9%.

EXAMPLE 12 preparation of crosslinked hemoglobin

The same preparation method as that of example 1 was conducted except that a static mixer was placed in the circulation line of the reaction system, and a branch line was provided at the static mixer to add the crosslinking agent, so that the hemoglobin and the crosslinking agent were sufficiently mixed at the static mixer, at a crosslinking agent addition rate of 100 mL/min.

The polymerized hemoglobin product of this example was tested using the second molecular weight distribution test method, and the results were: the MW >500Kd (< 10%) content is 8.1%, the MW <64Kd (< 5%) content is 4.6%.

EXAMPLE 13 preparation of crosslinked hemoglobin

Before polymerization, 0.5N sodium hydroxide solution is used for washing used pipelines, containers and accessories to remove pyrogens, injection water is used for washing the pipelines, the containers and the accessories to be removed to be neutral, a polymerization system is assembled according to a schematic diagram 2, particularly, the components of a static mixer 1 are assembled, the static mixer 1 is inserted into a circulating rubber tube 2, the tail end of a circulating hemoglobin flow direction 6 is bundled by a binding belt 7, the static mixer 1 is prevented from moving away when liquid flows, a liquid adding rubber tube 3 added with glutaraldehyde, a pH adjusting buffer solution and a sodium borohydride buffer solution is connected into the liquid adding rubber tube 3 through a T-shaped connector 4, a flow preventing clamp 5 is arranged on the liquid adding rubber tube 3 to prevent the backflow of the liquid, and after the assembly is completed, the high-. Assembling a polymerization system according to a schematic diagram 2, blowing nitrogen through a nitrogen circulating pipeline 11 and keeping the nitrogen till the polymerization is finished, calculating the amount of hemoglobin to be added, measuring and adding the hemoglobin into a polymerization reaction kettle 9, diluting the hemoglobin into 2g/dL by ultrapure water (injection water), adding NAC according to the proportion of 3mg/mL of final concentration, heating the diluted hemoglobin solution to 42 +/-2 ℃ through a heating jacket of the reaction kettle 9, taking the temperature according to the value of a reading thermometer 10, measuring glutaraldehyde according to the proportion of adding 37mg of 1g of hemoglobin, diluting the glutaraldehyde into 6.3mg/mL by ultrapure water (injection water), starting a circulating peristaltic pump 13 to uniformly mix the hemoglobin by the circulating hemoglobin and a magnetic stirrer 8 (the stirring speed is 150rpm), adding diluted glutaraldehyde 14 through a liquid adding peristaltic pump 12 within 50 minutes through a T-shaped connector 4, preserving heat for 20min after the polymerization is finished, then, the temperature is reduced to between 25 and 20 ℃, the pH of the hemoglobin polymerization solution is adjusted to 10 +/-0.2 by using a pH12.5 sodium borate buffer solution (borax 38g/L), sodium borohydride is weighed according to the proportion of 15.6g of hemoglobin, the sodium borohydride is diluted to 9.5mg/mL by using a pH10.5 sodium borate buffer solution (borax 4.6g/L), the sodium borohydride buffer solution is added within 15min through a T-shaped connector 4, the solution is changed to below the pH7.8 by using a 30KD ultrafiltration membrane pH7.6 disodium hydrogen phosphate solution changing buffer solution, and then the solution is changed to a pH7.6 dialysis buffer solution C (6.7g/L sodium chloride, 0.3g/L potassium chloride, 0.15g/L calcium chloride, 2.0g/L N-acetylcysteine and 3.1g/L sodium lactate) to obtain the polymerized hemoglobin.

The polymerized hemoglobin product of this example was tested using the first molecular weight distribution test method, and the results were: the dimer content was 3.96%, the tetramer plus octamer content was 63.53%, and the high polymer content was 32.51%, with the results shown in FIG. 3.

EXAMPLE 14 preparation of crosslinked hemoglobin

Before polymerization, 0.5N sodium hydroxide solution is used for washing used pipelines, containers and accessories to remove pyrogens, injection water is used for washing the pipelines, the containers and the accessories to be removed to be neutral, a polymerization system is assembled according to a schematic diagram 2, particularly, the components of a static mixer 1 are assembled, the static mixer 1 is inserted into a circulating rubber tube 2, the tail end of a circulating hemoglobin flow direction 6 is bundled by a binding belt 7, the static mixer 1 is prevented from moving away when liquid flows, a liquid adding rubber tube 3 added with glutaraldehyde, a pH adjusting buffer solution and a sodium borohydride buffer solution is connected into the liquid adding rubber tube 3 through a T-shaped connector 4, a flow preventing clamp 5 is arranged on the liquid adding rubber tube 3 to prevent the backflow of the liquid, and after the assembly is completed, the high-. Assembling a polymerization system according to a schematic diagram 2, blowing nitrogen through a nitrogen circulating pipeline 11 and keeping the nitrogen till the polymerization is finished, calculating the amount of hemoglobin to be added, measuring and adding the hemoglobin into a polymerization reaction kettle 9, diluting the hemoglobin to be 3g/dL by ultrapure water (injection water), adding NAC according to the proportion of 3mg/mL of final concentration, heating the diluted hemoglobin solution to 42 +/-2 ℃ through a heating jacket of the reaction kettle 9, taking the temperature based on reading the value of a thermometer 10, measuring glutaraldehyde according to the proportion of 45mg added to 1g of hemoglobin, diluting the glutaraldehyde to be 6.3mg/mL by the ultrapure water (injection water), starting a circulating peristaltic pump 13 to uniformly mix the hemoglobin by the circulating hemoglobin and a magnetic stirrer 8 (the stirring speed is 150rpm), adding diluted glutaraldehyde 14 through a liquid adding peristaltic pump 12 within 50 minutes through a T-shaped connector 4, preserving heat for 20min after the polymerization is finished, then, the temperature is reduced to between 25 and 20 ℃, the pH of the hemoglobin polymerization solution is adjusted to 10 +/-0.2 by using a pH12.5 sodium borate buffer solution (borax 38g/L), sodium borohydride is weighed according to the proportion of 15.6g of hemoglobin, the sodium borohydride is diluted to 9.5mg/mL by using a pH10.5 sodium borate buffer solution (borax 4.6g/L), the sodium borohydride buffer solution is added within 15min through a T-shaped connector 4, the solution is changed to below the pH7.8 by using a 30KD ultrafiltration membrane pH7.6 disodium hydrogen phosphate solution changing buffer solution, and then the solution is changed to a pH7.6 dialysis buffer solution C (6.7g/L sodium chloride, 0.3g/L potassium chloride, 0.15g/L calcium chloride, 2.0g/L N-acetylcysteine and 3.1g/L sodium lactate) to obtain the polymerized hemoglobin.

The polymerized hemoglobin product of this example was tested using the first molecular weight distribution test method, and the results were: the dimer content was 2.68%, the tetramer plus octamer content was 58.93%, and the polymer content was 38.40% (each data set was rounded up, possibly resulting in a less than 100% additive result), as shown in FIG. 4.

EXAMPLE 15 preparation of crosslinked hemoglobin

Before polymerization, 0.5N sodium hydroxide solution is used for washing used pipelines, containers and accessories to remove pyrogens, injection water is used for washing the pipelines, the containers and the accessories to be removed to be neutral, a polymerization system is assembled according to a schematic diagram 2, particularly, the components of a static mixer 1 are assembled, the static mixer 1 is inserted into a circulating rubber tube 2, the tail end of a circulating hemoglobin flow direction 6 is bundled by a binding belt 7, the static mixer 1 is prevented from moving away when liquid flows, a liquid adding rubber tube 3 added with glutaraldehyde, a pH adjusting buffer solution and a sodium borohydride buffer solution is connected into the liquid adding rubber tube 3 through a T-shaped connector 4, a flow preventing clamp 5 is arranged on the liquid adding rubber tube 3 to prevent the backflow of the liquid, and after the assembly is completed, the high-. Assembling a polymerization system according to a schematic diagram 2, blowing nitrogen through a nitrogen circulating pipeline 11 and keeping the nitrogen till the polymerization is finished, calculating the amount of hemoglobin to be added, measuring and adding the hemoglobin into a polymerization reaction kettle 9, diluting the hemoglobin to be 1g/dL by ultrapure water (injection water), adding NAC according to the proportion of 1mg/mL of final concentration, heating the diluted hemoglobin solution to 42 +/-2 ℃ through a heating jacket of the reaction kettle 9, taking the temperature based on reading the value of a thermometer 10, measuring glutaraldehyde according to the proportion of 29mg added to 1g of hemoglobin, diluting the glutaraldehyde to be 6.3mg/mL by the ultrapure water (injection water), starting a circulating peristaltic pump 13 to uniformly mix the hemoglobin by the circulating hemoglobin and a magnetic stirrer 8 (the stirring speed is 150rpm), adding diluted glutaraldehyde 14 through a liquid adding peristaltic pump 12 within 50 minutes through a T-shaped connector 4, preserving heat for 20min after the polymerization is finished, then, the temperature is reduced to between 25 and 20 ℃, the pH of the hemoglobin polymerization solution is adjusted to 10 +/-0.2 by using a pH12.5 sodium borate buffer solution (borax 38g/L), sodium borohydride is weighed according to the proportion of 15.6g of hemoglobin, the sodium borohydride is diluted to 9.5mg/mL by using a pH10.5 sodium borate buffer solution (borax 4.6g/L), the sodium borohydride buffer solution is added within 15min through a T-shaped connector 4, the solution is changed to below pH7.8 by using a 30KD ultrafiltration membrane pH7.6 disodium hydrogen phosphate solution changing buffer solution, and then the solution is changed to a pH7.6 dialysis buffer solution C (6.7g/L sodium chloride, 0.3g/L potassium chloride, 0.15g/L calcium chloride, 2.0g/L N-acetylcysteine and 3.1g/L sodium lactate) to obtain the polymerized hemoglobin.

The polymerized hemoglobin product of this example was tested using the first molecular weight distribution test method, and the results were: dimer content 4.24%, tetramer plus octamer content 68.08%, and high polymer content 27.68% (each data is rounded off, possibly resulting in a less than 100% additive result) with specific results shown in FIG. 5.

EXAMPLE 16 preparation of crosslinked hemoglobin

The same preparation method as in example 13 was conducted except that glutaraldehyde was measured in a ratio of 1g hemoglobin to 29mg before diluting glutaraldehyde to 6.3mg/mL with ultrapure water (water for injection), that is, the mass ratio of hemoglobin to glutaraldehyde was 1000: 29; meanwhile, hemoglobin was diluted to 3g/dL with ultrapure water (water for injection).

The polymerized hemoglobin product of this example was tested using the first molecular weight distribution test method, and the results were: dimer content 4.73%, tetramer plus octamer content 67.15%, and polymer content 28.12% (each data set is rounded up, possibly resulting in a result that adds up to less than 100%).

EXAMPLE 17 preparation of crosslinked hemoglobin

The same preparation method as in example 13 was conducted except that glutaraldehyde was measured in a ratio of 45mg to 1g of hemoglobin before diluting glutaraldehyde to 6.3mg/mL with ultrapure water (water for injection), that is, the mass ratio of hemoglobin to glutaraldehyde was 1000:45, a first step of; meanwhile, hemoglobin was diluted to 1g/dL with ultrapure water (water for injection).

The polymerized hemoglobin product of this example was tested using the first molecular weight distribution test method, and the results were: dimer content 4.16%, tetramer plus octamer content 66.69%, and polymer content 29.15% (each data is rounded off, possibly resulting in a less than 100% addition).

EXAMPLE 18 preparation of Cross-Linked hemoglobin

The same procedure as in example 13 was followed, except that diluted glutaraldehyde was added via a T-connector over 40 minutes by means of an addition peristaltic pump, before 20min of the incubation cycle.

The polymerized hemoglobin product of this example was tested using the first molecular weight distribution test method, and the results were: dimer content 4.45%, tetramer plus octamer content 64.93%, polymer content 30.62% (each data set is rounded up, possibly resulting in a result that adds up to less than 100%).

EXAMPLE 19 preparation of crosslinked hemoglobin

The same procedure as in example 13 was followed, except that diluted glutaraldehyde was added via a T-connector over a period of 60 minutes by means of an addition peristaltic pump, before the incubation cycle was repeated for 20 min.

The polymerized hemoglobin product of this example was tested using the first molecular weight distribution test method, and the results were: dimer content 3.78%, tetramer plus octamer content 62.01%, high polymer content 34.21% (each data is rounded off, possibly resulting in an additive other than 100%).

Comparative example 1 Effect of adding no NAC on the molecular weight distribution of Cross-Linked hemoglobin

The same procedure as in example 1 was followed, except that N-acetyl-L-cysteine (NAC) was not added to the diluted deoxyhemoglobin solution before the addition of the crosslinking agent, and the molecular weight distribution of the resulting crosslinked hemoglobin was measured by the above-mentioned molecular weight distribution measuring method two, and is shown in Table 1.

Comparative example 2 Effect of glutathione in place of NAC on Cross-Linked hemoglobin molecular weight distribution

The same procedure as in example 1 was followed, except that glutathione was added to the diluted deoxyhemoglobin solution before the addition of the crosslinking agent to a final concentration of 3mg/mL, and the molecular weight distribution of the resulting crosslinked hemoglobin was measured by the above-mentioned molecular weight distribution measuring method two, and is shown in Table 1.

Comparative example 3 Effect of mercaptoethylamine instead of NAC on the molecular weight distribution of crosslinked hemoglobin

The same procedure as in example 1 was followed, except that mercaptoethylamine was added to the diluted deoxyhemoglobin solution before the addition of the crosslinking agent at a final concentration of 3mg/mL, and the molecular weight distribution of the resulting crosslinked hemoglobin was determined by the above-mentioned molecular weight distribution measuring method two, as shown in Table 1.

TABLE 1

The results in table 1 show:

when glutaraldehyde is crosslinked, NAC is added to remarkably improve the intermolecular crosslinking of hemoglobin, and the mechanism of the method is that NAC inhibits the formation of disulfide bonds of hemoglobin, so that the disulfide bonds are uniformly distributed in a solution, the formation of protein with uniform molecular weight is facilitated, the residue of unpolymerized low-molecular-weight hemoglobin is reduced, and the formation of a high-molecular-weight hemoglobin polymer is reduced.

Comparative example 4 Effect of concentration ratio of hemoglobin to NAC on the molecular weight distribution of Cross-Linked hemoglobin

The same preparation method as in example 1, except that the concentration of NAC in the solution is 0.2mg/mL, i.e. the ratio of the concentration of hemoglobin to NAC is 100: 1, the molecular weight distribution of the cross-linked hemoglobin obtained by the above-mentioned molecular weight distribution detection method II is shown in Table 2.

TABLE 2

The results in table 2 show:

the effect of adding different concentrations of NAC on the degree of crosslinking before adding glutaraldehyde for crosslinking is significantly different, as compared to the hemoglobin: the NAC mass ratio is in the range of 5-10:1, the effect is better, when the added NAC concentration is too low (for example, the mass ratio of the hemoglobin to the NAC is 100: 1), the content of small molecules in the cross-linked hemoglobin is too high, which exceeds 5%, and the quality standard of molecular weight distribution is not met.

Comparative example 5 Effect of the Mass ratio of hemoglobin to glutaraldehyde on the molecular weight distribution of Cross-Linked hemoglobin

The same preparation method as in example 13 was conducted except that glutaraldehyde was measured in a proportion of 50mg to 1g of hemoglobin before diluting glutaraldehyde to 6.3mg/mL with ultrapure water (water for injection), that is, the mass ratio of hemoglobin to glutaraldehyde was 1000: 50, the molecular weight distribution of the crosslinked hemoglobin obtained by the above first method of molecular weight distribution is shown in Table 3.

Comparative example 6 Effect of the Mass ratio of hemoglobin to glutaraldehyde on the molecular weight distribution of Cross-Linked hemoglobin

The same preparation method as in example 13 was conducted except that glutaraldehyde was measured in a ratio of 1g hemoglobin to 24mg before diluting glutaraldehyde to 6.3mg/mL with ultrapure water (water for injection), that is, the mass ratio of hemoglobin to glutaraldehyde was 1000: 24, the molecular weight distribution of the crosslinked hemoglobin obtained by the first molecular weight distribution measurement method is shown in Table 3.

Comparative example 7 Effect of diluted concentration of hemoglobin on molecular weight distribution of crosslinked hemoglobin

The procedure of example 13 was repeated, except that 0.5g/dL of hemoglobin was diluted with ultrapure water (water for injection), and the molecular weight distribution of the resulting crosslinked hemoglobin was measured by the first molecular weight distribution measuring method described above, and the molecular weight distribution was as shown in Table 3.

Comparative example 8 Effect of diluted concentration of hemoglobin on molecular weight distribution of crosslinked hemoglobin

The same procedure as in example 13 was repeated, except that hemoglobin was diluted with ultrapure water (water for injection) to 4g/dL, and the molecular weight distribution of the crosslinked hemoglobin was measured by the first molecular weight distribution measuring method described above, and the molecular weight distribution of the crosslinked hemoglobin was measured as shown in Table 3.

TABLE 3

The results in table 3 show:

comparative example 5 shows the results in hemoglobin: when the proportion of the glutaraldehyde is less than 1000:45, namely when the addition amount of the glutaraldehyde is too high, the content of the high polymer is higher;

comparative example 6 shows the results in hemoglobin: the proportion of glutaraldehyde is more than 1000: when 29 hours are needed, namely when the addition amount of the glutaraldehyde is too low, the dimer content is higher;

comparative example 7 shows that when the dilution of hemoglobin is less than 1g/dL, although the molecular weight distribution is within the acceptable range, the lower dilution is not recommended in view of the larger polymerization volume required for the lower dilution and the higher production cost;

comparative example 8 shows that the polymer content is higher at a hemoglobin dilution of greater than 3 g/dL;

examples 13-17 show that the molecular weight distribution is more desirable when the glutaraldehyde addition ratio (hemoglobin: glutaraldehyde) is 1000:29-45 and the hemoglobin dilution is 1-3 g/dL.

Comparative example 9 Effect of polymerization temperature on molecular weight distribution of crosslinked hemoglobin

The same procedure as in example 13 was conducted, except that the diluted hemoglobin solution was heated to 30 ℃ by the heating mantle of the reaction vessel, and the molecular weight distribution of the resulting crosslinked hemoglobin was measured by the first molecular weight distribution measuring method described above, and the molecular weight distribution thereof was as shown in Table 4.

Comparative example 10 Effect of agitation Rate on crosslinked hemoglobin molecular weight distribution

The same procedure as in example 13, except that the stirring rate was 250rpm, the molecular weight distribution of the crosslinked hemoglobin was measured by the above-mentioned first method of measuring molecular weight distribution, and the molecular weight distribution of the crosslinked hemoglobin was found to be shown in Table 4.

Comparative example 11 Effect of polymerization time on molecular weight distribution of crosslinked hemoglobin

The same procedure as in example 13 was followed, except that diluted glutaraldehyde was added through a T-connector within 30 minutes by means of a liquid adding peristaltic pump before the incubation cycle was carried out for 20min, and the molecular weight distribution of the resulting crosslinked hemoglobin was measured by the above-mentioned first molecular weight distribution measuring method, and is shown in Table 4.

Comparative example 12 Effect of polymerization time on molecular weight distribution of crosslinked hemoglobin

The same procedure as in example 13 was followed, except that diluted glutaraldehyde was added through a T-connector for 70 minutes by a liquid adding peristaltic pump before the incubation cycle was carried out for 20 minutes, and the molecular weight distribution of the resulting crosslinked hemoglobin was measured by the above-mentioned first molecular weight distribution measuring method, and is shown in Table 4.

TABLE 4

The results in table 4 show:

the comparison of comparative example 9 with example 13 shows that the effect on the degree of polymerization is not significant when only the polymerization temperature is changed, and the present invention continues to use 42. + -.2 ℃ in consideration of the polymerization temperature of the original product;

the results of comparing comparative example 10 with example 13 show that the effect on the polymerization is not significant when only the stirring rate of the reaction vessel is changed, and it is recommended to use a lower rotation speed in consideration of the possibility of causing foaming of the solution at a higher rotation speed;

comparative examples 11, 12 and examples 13, 18, 19 show that the time, which affects the degree of polymerization more significantly, is compared with the different polymerization times, and that the dimer content is higher below 40 minutes, and that the dimer content is not significantly reduced but the polymer content is higher above 60 minutes, so that the time for adding glutaraldehyde is suitably selected to be 40 to 60 minutes, preferably 50 minutes.

Comparative example 13 comparison of the product of the present invention with oxyglobin, a product originally developed by biopure Co

The comparison results between the product of the present invention and the oxyglobin of the product originally developed by biopure corporation, measured by the first molecular weight distribution method, are shown in Table 5 (each data is rounded up, which may result in a result that the sum is not 100%).

TABLE 5

The results in table 5 show:

the product polymerized within the polymerization parameter range provided by the invention is 10 percent lower than the method provided by the patent number 99808286.4 and the polymer of the only glutaraldehyde polymerized hemoglobin product on the market, which shows that the invention has obvious improvement effect.

In summary, the above comparative experiments show that:

the addition ratio of glutaraldehyde is 1000:29-45, the dilution concentration of hemoglobin is 1-3g/dL, and the concentration ratio of hemoglobin to NAC is 5-10:1 is a preferable polymerization condition.

Preparation of organ perfusate

EXAMPLE 20 preparation of organ perfusate

1. Weighing the components of the solution I according to the preparation proportion of each 1000mL of organ perfusate:

human albumin (Shandongtai bang biology, pharmaceutical grade) 50g

Heparin (Chenxin pharmaceutical industry) 10000IU

10mL of 8.4% sodium bicarbonate (Tiandi pharmacy)

10mL of 10% calcium gluconate (Huarun double crane)

Vancomycin (North China pharmaceutical) 0.5mg

Gentamicin (Huarui pharmaceutical) 0.06mg

50mL of 10% compound amino acid injection (Huarui pharmaceutical)

0.2mL of composite vitamin preparation of Schnivista (Pude pharmaceutical industry)

Vitamin K1 (honest pharmaceutical industry) 0.1mg

Adding human albumin, heparin, 8.4% of sodium bicarbonate, 10% of calcium gluconate, vancomycin, gentamicin, 10% of compound amino acid injection, a Schnivitamin complex preparation (namely 12 complex vitamins for injection), vitamin K1 and distilled water according to the proportion of the formula, quantifying, filtering, sterilizing, aseptically filling and sealing to obtain a solution I;

2. weighing the components of the solution II according to the preparation proportion of each 1000mL of organ perfusate:

30g of the crosslinked hemoglobin obtained in example 1 was sampled

The sterile filling distribution system is filled in a sterile and oxygen-free environment, the filled medicine bag is required to be sterile, and the oxygen isolation rate is less than 0.008cc/m²24hrs, replacing residual air in the medicine bag by introducing inert gas for many times before filling, filling a solution containing 30g of cross-linked hemoglobin, and sealing by a thermoplastic method to obtain a solution II;

3. providing a solution III component according to the proportion of organ perfusate per 1000 mL:

providing 16mL of separately packaged prostacyclin (2ug/mL, Mingren medicine) according to a formula ratio, and separately and continuously infusing when in use, wherein the infusion speed is 4 mL/hr;

the solution I and the solution II are mixed in proportion before use and perfused by matching with the solution III to obtain the organ perfusate.

After the organ perfusate prepared by the embodiment is stored for one year, the activity of the contained cross-linked hemoglobin is detected, the detection result is shown in table 6, and the methemoglobin and the oxyhemoglobin are both less than 5 percent and meet the quality requirement.

TABLE 6

EXAMPLE 21 preparation of organ perfusate

adding human albumin, heparin, 8.4% of sodium bicarbonate, 10% of calcium gluconate, vancomycin, gentamicin, 10% of compound amino acid injection, a Schnivitamin complex preparation and vitamin K1 according to the formula proportion, quantifying distilled water, filtering for sterilization, aseptic filling and sealing to obtain a solution I;

50g of the crosslinked hemoglobin prepared in example 1 was taken

The distribution system used for sterilization and filling is used for completing filling in a sterile and oxygen-free environment, the filled medicine bag is required to be sterile, the oxygen separation rate is less than 0.008cc/m2/24hrs, residual air in the medicine bag is replaced by introducing inert gas for many times before filling, and after a solution containing 50g of cross-linked hemoglobin is filled, the solution II is sealed in a thermoplastic mode to obtain a solution II;

3. providing a solution III according to the proportion of organ perfusate per 1000 mL:

providing 32mL of separately packaged prostacyclin (2ug/mL, Mingren medicine) according to the formula proportion, and continuously infusing separately when in use, wherein the infusion speed is 6 mL/hr;

the solution I and the solution II are mixed in proportion before use, and perfused by matching with the solution III to obtain organ perfusate.

Thirdly, research on safety and effectiveness of canine kidney in-vitro perfusion

After the induction of canine anesthesia, the abdominal cavity was rapidly opened, both kidneys were excised, and the isolated kidneys were perfused with the organ perfusate prepared in example 20 for safety and efficacy studies of the organ perfusate.

1. Fluorescent dye labeling of the crosslinked hemoglobin prepared in example 1

Prior to the preparation of the organ perfusate of example 20, the cross-linked hemoglobin prepared in example 1 was first labeled with a fluorescent dye, preferably fluorescein isothiocyanate, by modifying the method described by Wilderspin in anal. biochem.,132:449(1982) and anal. biochem.,215:17-23(1993) to label the hemoglobin in the cross-linked hemoglobin solution with fluorescein isothiocyanate (hereinafter "FITC"). A FITC-labeled stock solution was prepared by dissolving 6.6g of FITC in 615mL of 100mM borate buffer (pH 9.5). 1850mL of the crosslinked hemoglobin solution obtained in example 1 was taken and reacted with stirring continuously at room temperature under a nitrogen atmosphere for 2 hours. Residual FITC was removed by diafiltration through a 30kD membrane, exchanged for seven volumes with a lactic acid storage solution (ph7.7), and then concentrated.

2. Investigation of safety and efficacy

Organ perfusate was prepared from the labeled cross-linked hemoglobin according to the method of preparing organ perfusate of example 20, and the kidney was perfused. After 72 hours, the tissue was washed with PBS buffer several times to wash out hemoglobin. Take 0.5x0.5x0.1cm³Dehydrating, waxing and slicing kidney tissues conventionally, performing HE staining, performing light microscope observation, judging activity change of the tissues according to change of tissue forms, and simultaneously observing whether hemoglobin is adhered to the tissues and whether hemoglobin exudes from kidney envelopes by using a fluorescence microscope; take 1cm³Kidney cortex tissue is pre-fixed for 2 hours at 4 ℃ by using 2.5% glutaraldehyde (0.1M) phosphate buffer solution with pH7.4, washed for 3 times in phosphate buffer solution with pH7.38 for 1 hour, dehydrated step by using 50%, 70%, 80%, 90% and 95% acetone for 15 minutes each time, finally embedded in a 60-DEG incubator for polymerization for 48 hours, an ultrathin section is prepared by an ultrathin slicer, double electron staining is carried out by uranyl acetate and lead citrate, and JEM-1200EX electron microscope observation is carried out to further observe the change of cell morphology and further judge the change of organ activity.

Fluorescence microscopy results showed that no adherent hemoglobin was found in the tissue after perfusion rinsing, nor was exuded hemoglobin observed in the kidney envelope; the results of the light microscope show that after 72 hours of perfusion, the renal tissue structure changes slightly, and clear renal tubules and glomeruli are visible under the low power microscope. Under the high power mirror, the glomerulus has clear outline and basically consistent size. Except for a small amount of mild renal proximal tubular swelling, most of the structures are normal; electron microscopy results show that after 72 hours of perfusion, the karyotype is approximately normal, the nucleoplasm is evenly distributed, the mitochondria are slightly swollen, but the cristae arrangement is good, and the individual cristae membranous gaps are slightly widened. There is also a slight swelling of the endoplasmic reticulum. The proximal tubular epithelial cells have microvilli edema, thickening and falling off individually. The capillary endothelium and the endothelial cells of the filtration membrane are slightly edematous and have clear structures. In conclusion, no tissue adhesion and exudation of hemoglobin was found, and the activity of the perfused organ remained essentially normal for 72 hours, indicating that the perfusate was safe and effective.

Detailed production process and detection result disclosure of cross-linked hemoglobin

More than 99% of the cells in blood are red blood cells. The primary function of red blood cells is to transport hemoglobin, which in turn carries oxygen from the lungs to the tissues, carrying CO₂Carried from the tissue to the lungs. Normal red blood cells contain about 34 grams of hemoglobin per 100mL of cells. Approximately 1.33mL of oxygen can be bound per gram of hemoglobin. In bovine blood, the concentration of hemoglobin (bHB) was 10.1g/dL, i.e., at a volume of 2.96L of blood, which is equivalent to 299g bHB. Thus, bovine blood is a viable option for large-scale hemoglobin recovery.

Separation system for protein purification

For example, in some embodiments, the isolation system used for protein purification is the carrCentritechUnifuge system (or equivalent) from Pneumatic ScaaleAngellus. The centrifuge system uses a disposable module that does not require gamma irradiation of CIP and SIP. All process contact surfaces are easy to install and can be replaced by 100% after each run. Low shear harvesting of mammalian and insect cells is possible and minimal reduction in recovery of recovered cells is achievable. Since the cells are not lysed, the production of cell debris in the centrifuge is minimized, making the centrifuge an excellent choice for cell recovery or focused clarification. The single centrifuge module is easily plumbed to your disposable bioreactor attachment. The unit is fully automatic with flexible cycle parameter input. The feed suspension was pumped gently to the module and the cells settled to the outer diameter while the clear supernatant was continuously drained. Once the module is full of batteries, the controller stops the rotor and discharges the batteries. This cycle is repeated until the bioreactor volume is treated.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use assays, and the screening and treatment methods of the present invention are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLE 22 Process and Process control for Small batch glutaraldehyde polymerized bovine hemoglobin

Blood sampling

Bovine blood was obtained from a farm of the Qingdao Runwei cattle industry Co., Ltd (fig. 9 is a real view of the farm). Animals were continuously observed through their documented health program. The method comprises the steps of collecting the bovine blood through jugular veins, firstly confirming whether internal identification ear tags and epidemic prevention ear tags equipped by an animal health disease epidemic prevention center are complete or not, simultaneously conforming to the health standards of cattle, and carrying out subsequent blood collection after meeting the requirements. As required, each adult cow can collect 3L of blood at most each time. Before blood sampling, excrement and urine in a cowshed are cleaned, and the cowshed and the cattle body are kept clean and sanitary so as to avoid polluting blood sampling parts and blood samples. Then, the cattle is driven into a retaining fence, an assistant retains the head of the cattle, the head is slightly extended forwards and tilted upwards and slightly deflects to the opposite side, the neck is slightly bent, the jugular vein is exposed, local hair is cut, an iodophor cotton ball is used for wiping and disinfecting the cattle along the jugular vein in the direction of the cattle body, an operator wears a protective article, slightly bends over and squats at the exposed side of the jugular vein, the left thumb or the index finger and the middle finger are used for pressing the venous duct slightly below (near the heart end) the jugular vein groove to promote the jugular vein to be angry, part of the cattle is not clear due to fat, the left index finger and the middle finger are used for clicking the jugular vein groove at the moment to beat, then the cattle is pressed, the parts with the right index finger and the middle finger are used for touching, and a fluctua. The 16# needle head is clamped by the thumb, the index finger and the middle finger of the right hand, the needle head is aligned to the external jugular vein, the needle head is quickly and vertically punctured towards the vein direction by means of wrist force, blood returns immediately when the needle head is punctured into the vein, and the flow speed clamp on the blood sampling bag is opened to enable the blood to flow into the blood sampling bag. If the needle is deflected away from the vein (no flashback is seen), the needle can be withdrawn or pulled slightly subcutaneously and re-inserted after the vein is identified. If the dripping amount of blood is small or the speed is slow, the middle and lower ends of the jugular vein can be pressed by fingers for a moment. In the blood collection process, the blood bag is put on a shaking table to fully mix the blood and the anticoagulant. After blood sampling, the left hand is loosened, the needle head is pulled out rapidly, and the needle hole is pressed by an iodine tincture cotton ball for hemostasis for a moment. After the blood bag is full (the cattle blood live view collected in figure 10), the flow rate clamp is compressed, and the blood bag is put into a refrigerator for storage after being slightly cooled.

Cell washing

The collected blood was washed according to the method shown in fig. 11. Within 24 hours, 3-5 liters (L) of collected blood were transferred to a single mobilus 5L flexible bag (T100) using a peristaltic pump. A50L sodium citrate solution (7.9g/L sodium chloride and 6.0g/L sodium citrate dihydrate) was prepared in a sterile mixing tank and depyrogenated through a 10kDa membrane filter into a 50L flexible bag (T101). The citrus ized blood was pumped into a static in-line mixer at a flow rate of 200mL/min while mixing with a sodium citrate solution at a flow rate of 280mL/min, and the mixture was directed into a continuous 0.6 μm and 0.4 μm depth filter membrane and into a 20L flexible bag (T102). When bag T102 contained 5L of filtered blood, the wash process began by adjusting the transmembrane pressure to 15psi by recirculation through a 0.2 μm hollow fiber membrane at a rate of 1L/min, allowing an average permeate flow rate of 300 mL/min. Cell washing was initiated by diafiltration, by pumping sodium citrate solution into bag T102 at a flow rate of 300mL/min and continued until the cells were washed with 7 volumes. The diafiltration permeate was directed into a 50L flexible waste bag (T103). Diafiltration was continued until an equivalent of 7 volumes of exudate were collected. Examples of components used in the cell washing process are listed in table 7 below.

TABLE 7

An alternative to this method is to use larger scale equipment to perform this step, or to install a centrifuge and perform the C500 step at 25L to enable the bag to be adapted to perform at 25L. The current settings were designed to limit the canister (bag size) to 50L to fit a removable rack.

Cell lysis

When the cells lyse due to a rapid decrease in osmotic pressure, hemoglobin is released from the bovine red blood cells. Cell lysis and sequential diafiltration were performed on 100kDa and 30kDa membranes as shown in FIG. 12. The anticoagulated whole blood was pumped into a static mixer at a flow rate of 250mL/min while water for injection was added at a flow rate of 250mL/min, and filled into a 10L flexible bag (T105). When T105 was filled with 2.0-2.5L of diluted whole blood, the permeate was introduced into a 5L flexible bag (T106) by starting recirculation through a 100kDa hollow fiber membrane cassette (F103) at a flow rate of 1000 mL/min. When 1.0-1.5L of permeate accumulated in T106, recirculation of F104 through the 30kDa membrane (F104) was started at a flow rate of 1000mL/min, the permeate was waste. When the whole blood volume (T102) is less than 250mL, pumps 104 and 105 are stopped. Diafiltration is then started by pumping WFI directly into T105 at a flow rate of, for example, 250mL/min and continued until the concentration of hemoglobin in the 100kDa permeate is less than 0.2mg/mL, corresponding to a diafiltration volume of about 25-30L. Some examples of procedures used for cell lysis are listed in Table 8 below.

TABLE 8

Deoxygenation of hemoglobin solutions

The hemoglobin solution was stabilized by removing oxygen and deoxygenated using the method shown in fig. 13 for storage as an intermediate. Initially, a hemoglobin solution was pumped through two liquid phase degassing membranes arranged in series at a flow rate of 500mL/min with a counter-current flow of nitrogen at 75 psi. Deoxygenation was continued until the dissolved oxygen reading was below 0.02 mg/mL. When sufficient deoxygenation was achieved, the hemoglobin solution was filtered into a 5L flexible bag by pumping 0.3. mu.M and two 0.22. mu.M depth filters. The filtered hemoglobin can be stored for up to 2 weeks before further processing. Examples of components used in the hemoglobin filtration-deoxygenation process are listed in table 9 below.

TABLE 9

Chromatography

Chromatography was used to further purify the hemoglobin solution and reduce non-specific blood cell components (method shown in fig. 14). This was performed using a cepstrum SCG chromatography system equipped with a GE Healthcare XK borosilicate column (5cm i.d. × 100cm long) packed with fast flow Q sepharose (GE Healthcare), the bed height being 70 ± 5 cm. Buffer was prepared using water for injection and filtered through a 10kDa membrane to further reduce pyrogen content. The buffer solution is (1) buffer solution A; 2.42g/L Tris base, adjusting to pH9.0 +/-0.1, and (2) buffer B; 6.05g/LTris base, adjusted to pH 7.0. + -. 0.1(3) buffer C; 2.42g/L Tris base, adjusted to pH 8.9. + -. 0.1.

Prior to the chromatography procedure, 5 complete cycles of buffering were performed through a freshly packed Q sepharose column. Chromatography was performed at a flow rate of 125 mL/min. Hemoglobin solution, 1L containing 130. + -.10 mg/mL hemoglobin, was loaded onto the column first, then a pH gradient was formed by adding equal volumes of buffer A and buffer B, and the protein eluted from the column was measured by UV absorbance at 280 nm. When the absorbance of the eluent was below 0.05Au, the column pH was increased by elution with 100% buffer B. When the absorbance reached 0.43Au, the hemoglobin fraction was collected in a 20L flexible bag (T111), and when the absorbance dropped below 0.05Au, the hemoglobin fraction was terminated. After eluting hemoglobin, 3L of buffer C was pumped through the column to elute tightly bound components.

The column was washed with 0.2N phosphoric acid between each chromatography run, followed by two full buffer cycles. If another run is not initiated within 24 hours, the column is stored in 0.2N phosphoric acid. Examples of components used in the chromatographic process are listed in table 10 below.

Watch 10

Deoxidation

The purified hemoglobin was deoxygenated to increase stability, as shown in fig. 15A, 15B. The purified fraction from the anion exchange chromatography step was concentrated to 10. + -.1 mg/mL and ultrafiltered through a 30kDa hollow fiber membrane (F110). When the desired hemoglobin concentration was reached, the purified hemoglobin was deoxygenated by passing it through two degassing membranes (F108, F109) arranged in series at a flow rate of 500mL/min under a counter-current flow of nitrogen at 75 psi. Deoxygenation was continued until the dissolved oxygen reading was below 0.02 mg/mL.

The deoxygenated purified hemoglobin was then diafiltered into 6 volumes of storage buffer by pumping through a 30kDa hollow fiber membrane (F110). The composition of the storage buffer was 2.63g/L trisodium phosphate dodecahydrate, 7.0g/L disodium phosphate heptahydrate, and 2.0g/L acetylcysteine. When the buffer exchange was complete, the solution was filtered into a 5L flexible bag (T113) by pumping through 0.5 μ M and two 0.22 μ M depth filters. The purified hemoglobin can be stored at room temperature (17-23 ℃) in a nitrogen glove box for up to 60 days and then further processed. Examples of components used in the deoxygenation process are listed in table 11 below.

TABLE 11

Polymerisation

Purified hemoglobin was polymerized by cross-linking with glutaraldehyde using the method shown in fig. 16. Purified hemoglobin (4-5L, 110g/L) was transferred from a storage tank (T113) to a 20L temperature controlled fluctuation bag (T603) under nitrogen pressure. Water for injection was pumped through the purified hemoglobin transfer line into T603 to reduce the hemoglobin concentration to 20 g/L. The temperature of the diluted hemoglobin solution was then raised to 42 ± 2 ℃. A glutaraldehyde solution with a concentration of 6.2g/L was prepared in a temperature-controlled fluctuation bag (T602) and heated to 42. + -. 2 ℃. The glutaraldehyde solution was pumped into T603 for a period of 50min until the ratio of glutaraldehyde to hemoglobin was about 0.037: 1. Glutaraldehyde is added through a static mixer (M601) in the recirculation loop to ensure rapid and uniform mixing with the hemoglobin solution. When the addition of glutaraldehyde is complete, the temperature of the reaction mixture is cooled to below 25 ℃ and the solution is concentrated to a hemoglobin concentration of 60-70g/L by diafiltration through a 30kDa hollow fiber membrane (F601).

The glutaraldehyde-crosslinked hemoglobin bond was stabilized by reduction with sodium borohydride, as shown in fig. 17. Sodium borohydride decomposes in aqueous solution at neutral pH to form hydrogen and sodium borate. Diafiltration of the polymerized hemoglobin with sodium borate buffer was performed to stabilize sodium borohydride and limit hydrogen gas formation. The borate buffer consisted of 4.58g/l sodium borate decahydrate and 0.91g/l sodium hydroxide.

The buffer was filtered through a 10kDa membrane to reduce pyrogen content and stored in a 20L flexible bag (T605). The borate buffer was first pumped through the recirculation loop at a flow rate of 1000mL/min into T603. At the same time, the polymerized hemoglobin solution was diafiltered by pumping through a 30kDa hollow fiber membrane at a flow rate of 250 mL/min. The borate addition flow rate was adjusted to a flow rate equal to the diafiltration permeation rate, about 250 mL/min. Diafiltration was continued with borate buffer until 3 times the volume of the polymerized hemoglobin solution was added.

The sodium borohydride solution consisted of 9.45g/l sodium borohydride, 4.58g/l sodium borate decahydrate and 0.91g/l sodium hydroxide. The solution was filtered through a 10kDa membrane to reduce pyrogen content and stored in a 2L flexible bag (T606). First a solution of sodium borohydride (0.6L) was pumped through the recirculation loop at a flow rate of 7mL/min into T603, the temperature of T603 being controlled below 25 ℃.

The stabilized polymeric hemoglobin solution was concentrated to 100. + -.5 g/L hemoglobin on a 30kD ultrafiltration membrane (F601). The polymeric hemoglobin was diafiltered by diafiltration with a 30kD ultrafiltration membrane (F601) against diafiltrate solution a (6.67g/L sodium chloride, 0.30g/L potassium chloride, 0.20g/L calcium chloride dihydrate, 0.445g/L sodium hydroxide, 2.02g/L N-acetyl-L-cysteine, 3.07g/L sodium lactate, pH 4.9-5.1) to remove the boron containing components (sodium borate/sodium borohydride) and reduce the pH to 8.0-8.4. Examples of components used in the polymerization process are listed in table 12 below.

TABLE 12

Sterile filtration

The final polymerized hemoglobin solution was filtered through a 0.5 μ M filter, a sterile grade 0.2 μ M filter and a second sterile grade 0.2M filter into a20 liter soft bag. Bulk storage bags were stored under nitrogen until use.

Final product soft bag filling

Referring to fig. 18, the filling apparatus without gas residue of the present embodiment includes a tank 1, an inert gas source 2, a vacuum pump 3, a T-shaped pipeline 4 and a filling bag 5. The T-shaped pipeline 4 is composed of a tank body section 41, an air source section 42 and a vacuum section 43, three free ends of the T-shaped pipeline 4 are respectively connected with the tank body 1, the inert gas source 2 and the vacuum pump 3, and meanwhile, a first stop switch 61, a second stop switch 62 and a third stop switch 63 are respectively arranged at the port positions of the three free ends. The tank body 1 is internally stored with filling liquid, and the filling bag 5 is communicated with the vacuum section 43.

Referring to fig. 18, the specific steps of the filling operation of the stroma-free hemoglobin product by using the filling apparatus without gas residue of the embodiment are as follows: step S1, closing the first and third cut-off switches 61 and 63, opening the second cut-off switch 62, and introducing inert gas into the T-shaped pipe 4 and the filling bag 5 through the gas source section 42 to fill the T-shaped pipe 4 and the filling bag 5 with the inert gas; step S2, closing the second cut-off switch 62, opening the third cut-off switch 63 and the vacuum pump 3, evacuating all inert gases in the T-shaped pipeline 4 and the filling bag 5 by using the vacuum pump 3, repeating the operation of the step S1 and the operation of the step S2 three times in sequence, and pumping the interior of the T-shaped pipeline 4 and the filling bag 5 to a vacuum state of more than 0.1 Mpa; step S3, turning on the first cut-off switch 61, turning off the second cut-off switch 62 and the third cut-off switch 63, so that the stroma-free hemoglobin product in the tank 1 flows into the filling bag 5 through the tank section 41 and the vacuum section 43, thereby completing the gas-residue-free filling operation of the stroma-free hemoglobin product, and the filling product is shown in fig. 19.

EXAMPLE 23 description of the preparation procedure and Process control for the batch preparation of glutaraldehyde-crosslinked bovine hemoglobin

The preparation process of the glutaraldehyde cross-linked bovine hemoglobin raw material drug comprises the following main steps:

blood sampling

Bovine blood was obtained from the farm of the Renwei Qingdao cattle industry Co. Animals were continuously observed through their documented health program. The method comprises the steps of collecting the bovine blood through jugular veins, firstly confirming whether internal identification ear tags and epidemic prevention ear tags equipped by an animal health disease epidemic prevention center are complete or not, simultaneously conforming to the health standards of cattle, and carrying out subsequent blood collection after meeting the requirements. As required, each adult cow can collect 3L of blood at most each time. Before blood sampling, excrement and urine in a cowshed are cleaned, and the cowshed and the cattle body are kept clean and sanitary so as to avoid polluting blood sampling parts and blood samples. Then, the cattle is driven into a retaining fence, an assistant retains the head of the cattle, the head is slightly extended forwards and tilted upwards and slightly deflects to the opposite side, the neck is slightly bent, the jugular vein is exposed, local hair is cut, an iodophor cotton ball is used for wiping and disinfecting the cattle along the jugular vein in the direction of the cattle body, an operator wears a protective article, slightly bends over and squats at the exposed side of the jugular vein, the left thumb or the index finger and the middle finger are used for pressing the venous duct slightly below (near the heart end) the jugular vein groove to promote the jugular vein to be angry, part of the cattle is not clear due to fat, the left index finger and the middle finger are used for clicking the jugular vein groove at the moment to beat, then the cattle is pressed, the parts with the right index finger and the middle finger are used for touching, and a fluctua. The 16# needle head is clamped by the thumb, the index finger and the middle finger of the right hand, the needle head is aligned to the external jugular vein, the needle head is quickly and vertically punctured towards the vein direction by means of wrist force, blood returns immediately when the needle head is punctured into the vein, and the flow speed clamp on the blood sampling bag is opened to enable the blood to flow into the blood sampling bag. If the needle is deflected away from the vein (no flashback is seen), the needle can be withdrawn or pulled slightly subcutaneously and re-inserted after the vein is identified. If the dripping amount of blood is small or the speed is slow, the middle and lower ends of the jugular vein can be pressed by fingers for a moment. In the blood collection process, the blood bag is put on a shaking table to fully mix the blood and the anticoagulant. After blood sampling, the left hand is loosened, the needle head is pulled out rapidly, and the needle hole is pressed by an iodine tincture cotton ball for hemostasis for a moment. After the blood bag is full (the cattle blood live view collected in figure 10), the flow rate clamp is compressed, and the blood bag is put into a refrigerator for storage after being slightly cooled.

Cell washing

The collected blood was washed according to the method shown in fig. 20. 15-20L of blood collected over 24 hours was transferred to a 20L back-up loop flexible bag (T100) using a peristaltic pump. 200L of sodium citrate solution (7.9g/L sodium chloride and 6.0g/L sodium citrate dihydrate) was prepared in a sterile mixing tank and depyrogenated through a 10kDa membrane filter into a 200L Ultra Low Density Polyethylene (ULDP) disposable bag (T101). The anticoagulated blood was pumped into a static in-line mixer at a flow rate of 500mL/min while adding a sodium citrate solution at a flow rate of 700mL/min, the mixture was passed sequentially through 0.6. mu.M and 0.4. mu.M filter membranes and introduced into a 50L ULDP disposable bag (T102). When bag T102 contained 10L of filtered blood, the wash process started to adjust the transmembrane pressure to 15psi by recirculating through 0.2 μ M hollow fiber membrane at a rate of 2L/min, allowing a mean permeate flow rate of 500 mL/min. Cell washing was initiated by diafiltration, with a sodium citrate solution pumped into bag T102 at a flow rate of 500mL/min, and continued until the cells were washed with 7 diafiltration volumes. The diafiltration permeate was introduced into a 200L ULDP disposable bag (T103). Diafiltration was continued until an equivalent volume of 7 blood exudates was collected.

Examples of parts used in the cell washing process are listed in the following table 13, and examples of parts used in the cell washing process tested are listed in the following table 14.

Watch 13

TABLE 14

Cell lysis

Red blood cells were separated from white blood cells and platelets by centrifugation, and hemoglobin was released from the red blood cells when the cells were lysed by a rapid decrease in osmotic pressure, as shown in fig. 21. The washed blood cells were pumped into a tube bowl centrifuge (C201) operated at a centrifugal force of 13, 500 × g. The red blood cells contained in the heavy phase are directed through a static mixer (M202) where they are diluted 2-fold with water for injection and enter a 20L GE Ready loop flexible bag (T202). When T202 was filled with at least 10L of diluted whole blood, recirculation was started through a 100kDa hollow fiber membrane (F201) at a flow rate of 1000 mL/min. The permeate was directed to a 20L GE Ready loop flexible bag (T203). When 15L of permeate accumulated in T203, recirculation through the 30kDa membrane (F202) was started at a flow rate of 1000 mL/min. The F202 permeate is spent. Diafiltration by 100kDa (F201) was continued until the concentration of hemoglobin in the permeate was below 0.2mg/mL, indicating that most of the released hemoglobin had been extracted. This corresponds to approximately 15-20 diafiltration volumes. The hemoglobin separated from the cell debris by 100kDa filtration was concentrated by membrane filtration of 30 kDa. The steps of 100kDa and 30kDa are performed in a continuous process. When the hemoglobin concentration is in the range of 90-10g/L, the 30kDa filtration is stopped.

Examples of the fractions used in the cell lysis process are listed in table 15 below, and examples of the test fractions used in the cell lysis process are listed in table 16 below.

Watch 15

TABLE 16

Deoxygenation of hemoglobin solutions

The hemoglobin solution was stabilized by removing oxygen and filtered using the method shown in fig. 22 for storage as an intermediate. Initially, a hemoglobin solution was pumped through two liquid phase degassing membranes arranged in series at a flow rate of 500mL/min with a counter-current flow of nitrogen at 75 psi. Deoxygenation was continued until the dissolved oxygen reading was below 0.02 mg/mL. When sufficient deoxygenation was achieved, the hemoglobin solution was filtered into 20L GE Ready Circuit Flexible Bag (T301) by pumping through 0.3. mu.M and two 0.22. mu.M filters. The filtered hemoglobin can be stored for up to 2 weeks before further processing.

Examples of the components used in the hemoglobin filtration-deoxygenation process are listed in table 17 below, and examples of the components used in the hemoglobin filtration-deoxygenation process tested are listed in table 18 below.

TABLE 17

Watch 18

Chromatography

Chromatography was used to further purify the hemoglobin solution and reduce non-specific blood cell components (method shown in fig. 23). This was performed using a cepstrum SCG chromatography system equipped with a GE Healthcare XK borosilicate column (5cm i.d. × 100cm long) packed with fast flow Q sepharose (GE Healthcare), the bed height being 70 ± 5 cm. Buffer was prepared using water for injection and filtered through a 10kDa membrane to further reduce pyrogen content. The buffer solution is (1) buffer solution A; 2.42g/L Tris base, adjusting to pH9.0 +/-0.1, and (2) buffer B; 6.05g/LTris base, adjusted to pH 7.0. + -. 0.1(3) buffer C; 2.42g/L Tris base, adjusted to pH 8.9. + -. 0.1.

Prior to the chromatography procedure, 5 complete cycles of buffering were performed through a freshly packed Q sepharose column. Chromatography was performed at a flow rate of 125 mL/min. Hemoglobin solution, 1L containing 130. + -.10 mg/mL hemoglobin, was loaded onto the column first, then a pH gradient was formed by adding equal volumes of buffer A and buffer B, and the protein eluted from the column was measured by UV absorbance at 280 nm. When the absorbance of the eluent was below 0.05Au, the column pH was increased by elution with 100% buffer B. When the absorbance reached 0.43Au, the hemoglobin fraction was collected in a 20L flexible bag (T405), and when the absorbance dropped below 0.05Au, the hemoglobin fraction was terminated. After eluting hemoglobin, 3L of buffer C was pumped through the column to elute tightly bound components.

The column was washed with 0.2N phosphoric acid between each chromatography run, followed by two full buffer cycles. If another run is not initiated within 24 hours, the column is stored in 0.2N phosphoric acid. Examples of components used in the chromatographic process are listed in table 19 below, and examples of components used in the chromatographic process are listed in table 20 below.

Watch 19

Watch 20

Deoxidation

The purified hemoglobin was deoxygenated to increase stability, as shown in fig. 24A and 24B. The purified fraction from the anion exchange chromatography step was concentrated to 11. + -.1 mg/mL by filtration through a 30kDa hollow fiber membrane (F503). When the desired hemoglobin concentration was reached, the purified hemoglobin was deoxygenated by passing it through two degassing membranes (F501, F502) arranged in series at a flow rate of 500mL/min, with a counter flow of 75psi of nitrogen. Deoxygenation was continued until the dissolved oxygen reading was below 0.02 mg/mL.

The deoxygenated purified hemoglobin was then diafiltered by 6 volumes of storage buffer by pumping through a 30kDa hollow fiber membrane (F503). The composition of the storage buffer was 2.63g/L trisodium phosphate dodecahydrate, 7.0g/L disodium phosphate heptahydrate, and 2.0g/L acetylcysteine. When the buffer exchange was complete, the solution was filtered into a 20L GE Ready Circuit disposable bag (T501) by pumping through a 0.5 μ M filter and two 0.22 μ M filters. The purified hemoglobin can be stored at room temperature (17-23 ℃) in a nitrogen glove box for up to 60 days and then further processed.

Examples of the components used in the deoxidation process are listed in table 21 below, and examples of the components used in the deoxidation process tested are listed in table 22 below.

TABLE 21

TABLE 22

Polymerisation

Purified hemoglobin was polymerized by cross-linking with glutaraldehyde using the method shown in fig. 25. Purified hemoglobin (4-5L, 110g/L) was transferred from storage tank T501) to a 20L temperature controlled fluctuation bag (T603) under nitrogen pressure. Water for injection was pumped through the purified hemoglobin transfer line into T603 to reduce the hemoglobin concentration to 20 g/L. The temperature of the diluted hemoglobin solution was then raised to 42 ± 2 ℃. A glutaraldehyde solution with a concentration of 6.2g/L was prepared in a temperature-controlled fluctuation bag (T602) and heated to 42. + -. 2 ℃. Glutaraldehyde solution was pumped into T603 for a period of 50min until the ratio of glutaraldehyde to hemoglobin was about 0.037: 1. Glutaraldehyde is added through a static mixer (M601) in the recirculation loop to ensure rapid and uniform mixing with the hemoglobin solution. When the addition of glutaraldehyde is complete, the temperature of the reaction mixture is cooled to below 25 ℃ and the solution is concentrated to a hemoglobin concentration of 60-70g/L by diafiltration through a 30kDa hollow fiber membrane (F601).

The glutaraldehyde-cross-linked hemoglobin chemical bond was stabilized by reduction with sodium borohydride, as shown in fig. 26. Sodium borohydride decomposes in aqueous solution at neutral pH to form hydrogen and sodium borate. Diafiltration of the polymerized hemoglobin with sodium borate buffer was performed to stabilize sodium borohydride and limit hydrogen gas formation. The borate buffer consisted of 4.58g/L sodium borate decahydrate and 0.91g/L sodium hydroxide. The above buffer was filtered through a 10kDa membrane to reduce pyrogen content and stored in a 20L flexible bag (T605). The borate buffer was first pumped through the recirculation loop at a flow rate of 250mL/min into T603. At the same time, the polymerized hemoglobin solution was diafiltered by pumping through a 30kDa hollow fiber membrane at a flow rate of 1000 mL/min. The borate addition flow rate was adjusted to a flow rate equal to the diafiltration permeation rate, about 250 mL/min. Diafiltration was continued with borate buffer until 3 times the volume of the polymerized hemoglobin solution was added.

The sodium borohydride solution consisted of 9.45g/l sodium borohydride, 4.58g/l sodium borate decahydrate and 0.91g/l sodium hydroxide. The solution was filtered through a 10kDa membrane to reduce pyrogen content and stored in a 2L flexible bag (T606). First a sodium borohydride solution (0.6L) was pumped through the recirculation loop at a flow rate of 7mL/min into T603, the temperature of T603 being controlled at 20. + -. 2 ℃. The borohydride reaction was continued for 60 minutes after all the solution was added.

The stabilized polymeric hemoglobin solution was concentrated to 100. + -.5 g/L hemoglobin on a 30kD ultrafiltration membrane (F601). The polymeric hemoglobin was diafiltered by diafiltration with a 30kD ultrafiltration membrane (F601) against diafiltrate solution a (6.67g/L sodium chloride, 0.30g/L potassium chloride, 0.20g/L calcium chloride dihydrate, 0.445g/L sodium hydroxide, 2.02g/L N-acetyl-L-cysteine, 3.07g/L sodium lactate, pH 4.9-5.1) to remove the boron containing components (sodium borate/sodium borohydride) and reduce the pH to 8.0-8.4. Examples of parts used in the polymerization process are listed in table 23 below, and examples of parts used in the polymerization process tested are listed in table 24 below.

TABLE 23

Watch 24

Sterile filtration

The final polymerized hemoglobin solution was filtered through a 0.5 μ M filter (F701), a 0.2 μ M sterile grade membrane filter (F702) and a second sterile grade 0.2 μ M membrane filter (F703) into a 20L GE Ready Circuit Soft bag (T701). Bulk storage bags were stored under nitrogen until use. A schematic of the sterile filtration process is shown in FIG. 27. Examples of components used in the sterile filtration process are listed below in table 25.

TABLE 25

Final product soft bag filling

Referring to fig. 18, the specific steps of the filling operation of the stroma-free hemoglobin product by using the filling apparatus without gas residue of the embodiment are as follows: step S1, closing the first and third cut-off switches 61 and 63, opening the second cut-off switch 62, and introducing inert gas into the T-shaped pipe 4 and the filling bag 5 through the gas source section 42 to fill the T-shaped pipe 4 and the filling bag 5 with the inert gas; step S2, closing the second cut-off switch 62, opening the third cut-off switch 63 and the vacuum pump 3, evacuating all inert gases in the T-shaped pipeline 4 and the filling bag 5 by using the vacuum pump 3, repeating the operation of the step S1 and the operation of the step S2 three times in sequence, and pumping the interior of the T-shaped pipeline 4 and the filling bag 5 to a vacuum state of more than 0.1 Mpa; step S3, turning on the first cut-off switch 61, and turning off the second cut-off switch 62 and the third cut-off switch 63, so that the stroma-free hemoglobin product in the tank 1 flows into the filling bag 5 through the tank section 41 and the vacuum section 43, thereby completing the gas-residue-free filling operation of the stroma-free hemoglobin product.

Preferably, in this embodiment, the inert gas source is a nitrogen source, and nitrogen with a high mass density and no pollution is used as the inert gas to perform gas replacement and evacuation operations on the stroma-free hemoglobin product filling device. Similarly, in other embodiments, other inert gases can be selected for emptying the filling device according to different products to be filled, so that no pollution is guaranteed and the products to be filled are not affected.

EXAMPLE 24 apparatus and Components for manufacturing and purification Process

The red blood cell purification process includes the use of a separation system, see image 28, and schematic 29. The separation system can better remove plasma components, platelets and partial white blood cells.

An example of an aggregate component is depicted as an image (FIG. 30). In this assembly, the effect of glutaraldehyde addition ratio, hemoglobin dilution, polymerization time on the degree of polymerization was tested, see examples 13, 14, 15.

The tomographic field image is seen as image 31. Two different loading amounts were optimized for the C800 chromatography system, and the column efficiency was measured before loading as shown in FIGS. 32 and 26, and the chromatography buffer parameters are shown in Table 27. The curve of the loading 1 tomographic data is shown in image 33, which is an overload loading image. A curve image 34 of the tomosynthesis 2 data. The SDS-PAGE purity of the fraction collected in chromatography 2 was found to be 99.1%, and the result was shown in image 35.

Watch 26

Item	Detection liquid	Column effect	Symmetry property
				Results	0.8MNaCl	6440	1.035

Watch 27

The filtrate from the primary filtration was subjected to ultrafiltration washing using sodium citrate buffer at about 7 times the initial feed volume via a 0.65 μ M hollow fiber ultrafiltration system, and an example of the components and an example of operation for the 0.65 μ M diafiltration process are shown in image 36 and schematic 37. The 100kDa diafiltration process comprises a retentate, a permeate and a diafiltration buffer, including the addition of diafiltration buffer directly in the retentate tube through a T-joint to a static mixer to improve the homogeneity of the retentate; comprises controlling the flow of the penetrating fluid by a peristaltic pump, preventing the formation of a gel layer and the reduction of the flow, and realizing the bridging of a large pilot scale; and includes a feed that passes briefly through a 40 c heat exchanger prior to entering the membrane, which promotes an increase in the proportion of transient dimer forms to improve diafiltration efficiency and yield, image 38 and schematic 39 show examples and operating examples of components for a 100KD diafiltration process. Image 40 and schematic 39 show examples of components and operational examples for a 30KD diafiltration concentration process. C500, C800, end products, etc. are all subjected to deoxidation treatment for long-term storage, and image 41 and schematic 42 show examples of components and operation examples for deoxidation treatment of deaerated films.

Example 25 study of hemoglobin index based on oxygen Carrier modification

Several batches of modified hemoglobin oxygen carriers were produced according to the present disclosure and analyzed according to standard test methods. The batch results are listed in tables 28-30 below.

Watch 28

Batch: OXP2019001

Watch 29

Batch: OXP2019002

Watch 30

Batch: OXP2019003

Example 26 study of hemoglobin stability based on oxygen Carrier modification

Several batches of modified hemoglobin oxygen carriers were produced according to the present disclosure and analyzed for changes in molecular weight distribution according to standard test methods. The batch results are listed in tables 31-34 below.

Watch 31

Batch: OXP007-1

Watch 32

Batch: OXP007-2

Watch 33

Batch: OXP008-2

Watch 34

Batch: OXP008-3

Example 27 CGMP production of oxygen Carrier based modified hemoglobin

Referring to fig. 43, a commercial scale production design. The primary production chambers are 101, 102, 103, 104, which are designed to meet class C specification requirements, and 101 chamber washes the collected red blood cells by diafiltration with a tangential flow filtration system or by centrifugation in a disposable centrifuge. The erythrocytes were then lysed by osmotic pressure and the hemoglobin was then filtered through a 100kD TFF membrane. The permeate was collected and concentrated through a 30kD TFF membrane. Once the hemoglobin is at the target concentration, the hemoglobin solution is filtered and sterilized by a 0.22 mu M filter membrane to a live bag of a sterile container, and the whole operation process is finished in a closed system so as to reduce the risk of overproof microorganisms and endotoxin. The 102 compartments are further purified primarily by dedicated ion exchange chromatography according to the present disclosure, with the eluate collected in a suitable container to limit and prevent oxygen and particulate exposure. Handling and connection by a pipe welder and appropriate containment vessel reduces all risks of exposure to the indoor environment. The material was concentrated on a 30kDa TFF membrane with a 3 Xchange of disodium hydrogen phosphate buffer, and the hemoglobin solution was then filtered through a 0.22. mu.M filter into pre-sterilized bags or containers for storage until further processing (no open system transfer). The 103 compartment is used primarily for hemoglobin glutaraldehyde polymerization, quenching reactions and process equipment using 30kD membrane exchange buffer. Each vessel in the polymerization system is also recirculated through the closed system hydrophobic gas exchange membrane to remove any oxygen introduced into the system by adding chemicals and buffers to the process. The final polymerized hemoglobin product was filtered through a 0.22 μ M filter into a pre-sterilized container or bag. And (4) transferring the final product to a 104 room for aseptic and anaerobic filling of the product after the final product is detected to be qualified. The 104 chambers can realize aseptic filling in an inert gas environment, the whole process is completed in a closed system, a safety alarm and an automatic inert gas closing valve system are arranged to ensure the safety of operators, and the finished products enter a finished product warehouse through a transfer window after filling is finished to wait for the detection and release of the finished products. With further reference to fig. 43, the 105 chamber is designed to meet class C specifications. The chamber will support the process production by formulating the buffers used in the process. The chemicals used in the buffer solution will be weighed in a safety hood to control the particles. The buffer will be supplied to the process through the wall by a pipe.

Room cleaning with disinfectants was performed daily on a pharmaceutical prescribed SOP. The room will be cleaned monthly with a sporicide or drift in response to environmental monitoring programs. The method will be performed by using a closed, pre-sterilized, single use system. Sampling will be performed on containers that have been welded to the piping on the system to maintain a closed system condition.

As shown in fig. 43, the production preparation chamber 106 is designed to meet the class C specification. The room will be used to prepare the assembly for use in the sterilization process. The room comprises pharmacopoeia-compliant purified water for rinsing the material and injected water for performing a final rinse of the components as required. The room will also include a double-leaf autoclave for sterilization to perform the sterilization and delivery functions.

As shown in fig. 43, the utility room 107 contains utilities for supporting facility functions. It includes air compressor, nitrogen/argon system, PW system, WFI system.

As shown in fig. 44, it is a negative layer warehouse, mainly used for safely storing materials used in the production process, and it includes a packaging material and consumable material chamber 001, a reagent chamber 002, a final product light inspection and packaging chamber 003, a finished product chamber 004, and a reagent and product retention chamber 005. Every room all can be according to requirement temperature regulation and humidity, and each room divides into certified products district and non-certified products district as required, and every room all is equipped with the entrance guard, and the talent that has the authority can get into.

As shown in fig. 45, is a two-level QC, r & d, utility room. The physical and chemical chamber 201, the precision instrument chamber 202 and the microorganism chamber 203 are QC quality inspection areas, 204 and 205 are hemoglobin product process optimization chambers, and an access control system is arranged at the division position of the chambers to prevent the intersection with personnel and articles of QC. 206 chamber is positioned to provide an air conditioning system for the process production area and the QC microbial chamber.

Source of raw materials

The starting material for this process is bovine blood collected from a controlled donor population.

Producing area

All animals were from china. China is a non-mad cow disease country, indicating that domestic animals in this country are unlikely to be infected with BSE agents, but there are also precautions to be taken. EDQM certificates were obtained from the cattle blood collection farm, see FIG. 46.

Method for avoiding cross-contamination risk

Whole bovine blood for processing is collected at slaughter or cattle farms in a controlled space. Animals from approved suppliers entered the draw area from the feeding area. All animals present in any collection will have complete documentation according to the group management program including source and feed status. After bleeding or exsanguination, the animals are removed from the blood collection room or area for further processing back into the herd management area or slaughter facility.

Isolation of animals

Individually identified cattle arriving at a collection station or slaughterhouse are controlled from a managed herd. In the first case, according to standard group management procedures, a lot of control will be given to dedicated blood collection areas before they are entered. Cattle enter through chutes which, in the case of slaughterhouses, direct the cattle directly to a collection area or stunning platform. The blood collection facility is separate from the primary blood draw (if a slaughterhouse) or collection facility of the designated facility.

Blood sampling

The accuracy and integrity of the support file and identification of each animal was verified prior to each collection and the animals were examined for any signs of disease. Blood collection was performed using a closed system. The animal (if bled) can be immobilized and if harvested at one time, a non-pneumatic tie down method can be used to stun. The collection at the slaughterhouse is never used, nor is a process known as "piping" used. If at the slaughterhouse, the chain shackle is placed around the back hoof immediately after stunning and the animal is lifted to an inverted position. An overhead conveyor system moves carcasses along the line to a collection platform. In the case of a slaughterhouse donor, an incision is made subcutaneously from the jaw to the entrance of the thorax; the skin is then retracted from the exposed sulcus by an elastic cord wrapped around the back of the neck.

Blood was collected in a closed manner using a stainless steel cannula needle inserted into the jugular vein near the vena cava. The sterile cannula connects the sterile trocar to a sterile stainless steel container or plastic bag prepared with a sodium citrate anticoagulant. Approximately 10-15 liters of blood was collected over a period of approximately 30-60 seconds. After blood collection, the trocar is removed and the vessel is sealed. The carcass is then removed from the dedicated supervised collection facility and then moved to the main slaughterhouse processing station without being returned. If the blood is collected in a harmless anticoagulant charged collection bag in a controlled volume animal management facility where the animal is exsanguinated for 2-5 liters, the animal will be restrained during the donor period.

Each collection container holds blood from a single animal. The unique number of each collection container is recorded and associated with the animal number from the unique animal ear tag. The ear number is also associated with a unique slaughter animal number, which is used for tracking the cattle through the packaging plant. The animals were then examined for evidence of disease or contamination by an inspector trained by personnel in the Ministry of agriculture. The examiner is supervised by a veterinarian trained by the department of agriculture. If an animal is for any reason trapped by the department of agriculture staff for further inspection, the blood from the animal is discarded at the slaughterhouse. The filled collection container may exit the facility and be placed in ice and loaded onto a truck for transport to a separation facility. Similar cataloging is performed if the donor population is managed, and the bags are collected and cooled for transport to the initial processing facility.

Possibility of contamination of the collected blood by other tissues

Because of the closed method of blood collection and the process of control and recording by using well-trained operators, the possibility of contamination by other tissues is minimal. In slaughterhouses, the trachea and oesophagus are avoided by positioning the cutting edge of the trocar towards the blood vessels.

The location on the skull where the animal was stunned was physically distant from the location (1 meter) where the trocar was inserted. Any fluid or bone fragments from the stunning site cannot come into contact with the blood collection site due to the suspended position of the animal during blood collection. The collected blood does not contact the brain, spinal cord, eye, ileum, lymph node, proximal colon, spleen, tonsil, dura, pineal gland, placenta, cerebrospinal fluid, pituitary, adrenal gland, distal colon, nasal mucosa, peripheral nerve, bone marrow, liver, lung or pancreas. In addition, any potentially contaminating tissue will be removed during the factory blood collection process, wherein the blood is filtered sequentially through an 800 μ strainer, a 50 μ strainer, and a 60 depth filter. The 60 μ depth filter has a broad pore size distribution; the maximum pore size is 60 μ or microns.

Water system

Water for injection is produced by condensing pure steam into a 2000L storage tank maintained above 75 ℃, which is recirculated through the spray ball during operation to flush all internal surfaces. The hot loop does not have any direct point of use, but provides a cold loop that is recirculated through a heat exchanger to reduce the temperature to 25 ℃. One point of use is buffer preparation and the other is component preparation for final rinsing prior to sterilization in an autoclave. The cold circuit is sterilized by night hot water for a defined period of time.

The raw materials were stored at controlled room temperature except for the purified hemoglobin solution stored at 2-8 ℃. Standard single use product contact materials such as polypropylene, polycarbonate, silicone tubing, C-flex tubing and bags with an inert inner layer made of ultra low density polyethylene or equivalent are used for storage. The system will be flushed to remove particles prior to use and tested for leaks prior to disposal. If hygiene is required, the system is flushed with 0.5M NaOH for a specified time frame, then the NaOH is flushed out of the system and it is ensured that the residue is neutralized before disposal. The final product was stored at controlled room temperature.

Heating, ventilation, air conditioning (HVAC) and air treatment

The HVAC system provides HEPA filtered air to a clean room that has been cooled to reduce moisture to less than 60% relative humidity and reheated to a temperature required for operator comfort. The system is designed to have a sufficient rate of air change that is appropriate for a classification with a pressure cascade of 0.05 "between rooms of different classifications, where the main processing zone is at the highest pressure. The process kit is designed with an airlock to allow personnel and material changeover to be performed with minimal impact on the process area. The room was cleaned with approved sanitary agents according to standard operating procedures. Environmental monitoring of live and non-live particles is performed periodically based on room classification. Surface monitoring will also be performed in defined positions defined by standard operating procedures.

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims.

Claims

1. A method for preparing cross-linked hemoglobin, comprising the steps of:

2. a method for preparing cross-linked hemoglobin, comprising the steps of: